WO2018107684A1 - Method for treating and preventing atherosclerosis and complications thereof - Google Patents

Abstract

A method for treating and preventing atherosclerosis, comprising administering a preventively effective amount of plasminogen to a subject prone to suffer atherosclerosis. Also provided are a medicine, pharmaceutical composition, product, and kit comprising plasminogen and used for treating and preventing atherosclerosis.

Description

Method for preventing atherosclerosis and its complications Technical field

The present invention relates to a method for preventing and/or treating a disorder of fat metabolism and a related disorder thereof, comprising administering an effective amount of plasminogen to a subject susceptible to or suffering from a disorder of fat metabolism and a related disorder thereof to reduce fat Abnormal deposition of tissues and organs in the body, thereby achieving the purpose of preventing and/or treating disorders of fat metabolism and related disorders and complications.

Background technique

Fat metabolism disorder, also known as lipodystrophy, is one of the metabolic diseases, which is caused by primary or acquired factors, lipids (lipids) and their metabolic substances and amounts in blood and other tissues and organs. abnormal. Lipid metabolism involves digestion and absorption of lipids in the small intestine, entry into the blood circulation by the lymphatic system (transport by lipoproteins), transformation by the liver, storage in adipose tissue, and utilization by tissues when needed. The main function of lipids in the body is to oxidize energy. Adipose tissue is the energy store of the body. Fat can also protect the internal organs with skin, bones and muscles, prevent body temperature from spreading and help the absorption of fat-soluble vitamins in food. Phospholipids are an important structural component of all cell membranes, and cholesterol is a precursor of bile acids and steroid hormones (adrenal cortical hormones and gonadal hormones). Lipid metabolism is regulated by genetics, neurohumoral fluids, hormones, enzymes, and tissues such as the liver. When these factors are abnormal, it can cause lipid metabolism disorder and pathophysiological changes of related organs, such as hyperlipoproteinemia and its clinical syndrome, obesity, fatty liver and so on.

Hyperlipoproteinemia is caused by excessive lipoproteins in the blood. Lipids in the blood such as triglyceride (TG), free cholesterol (FC), cholesterol (CE) and phospholipids are rarely soluble in water, and only a macromolecular complex (lipoprotein) is formed with apolipoprotein (APO). In order to dissolve, operate and metabolize in the blood. When the blood lipid is higher than the upper limit of normal people, it is hyperlipemia. Hyperlipidemia is also called hyperlipoproteinemia because blood lipids are transported in the form of lipoproteins in the blood. Generally, adult fasting blood triglycerides exceed 160mg/dl, cholesterol exceeds 260mg/dl, and children's cholesterol exceeds 160mg/dl as standard ^[1] .

Hyperlipoproteinemia (hyperlipidemia) is one of the important causes of atherosclerotic lesions and is an abnormal manifestation of lipid metabolism in the body. Due to the different types of blood lipids or lipoproteins, the types of blood lipids or lipoproteins outside the normal range may also be different. Therefore, the World Health Organization (WHO) classifies hyperlipoproteinemia into five types: type I, mainly chylomicrons, serum. The turbidity is milky white, which contains a large amount of triglyceride (TG); the type II is divided into two subtypes IIa and IIb. The former is mainly a low-density lipoprotein (LDL), and the latter is very low-density lipoprotein (VLDL). There is also an increase; type III, serum turbidity LDL and VLDL are increased, electrophoresis on the fusion; type IV, mainly VLDL increase, serum or turbidity; V type, chylomicrons and VLDL increase, serum opacity is milky white . Among them, type II and type IV are the most common ^[1] .

Hyperlipidemia can be divided into primary and secondary categories according to the cause. Primary is mostly caused by congenital defects (or genetic defects) in lipid and lipoprotein metabolism, as well as certain environmental factors (including diet, nutrition, and drugs) through unknown mechanisms. Secondary is mainly secondary to certain diseases such as diabetes, liver disease, kidney disease, thyroid disease, as well as alcohol and obesity. Environmental factors such as diet and lifestyle are also the cause of the disease.

Diabetes often involves disorders of lipid metabolism, so diabetes is also known as "glycolipidosis" ^[2] . The pathogenesis of diabetes is related to B cell function damage and insulin resistance, which is characterized by chronic hyperglycemia, and the disorder of glucose metabolism often involves disorder of lipid metabolism. Diabetic lipid metabolism disorder has become an independent risk factor for cardiovascular disease, mainly characterized by hypertriglyceridemia, low levels of HDL, and increased LDL concentrations.

The mechanism of diabetes lipid metabolism disorder is still unclear, but there is a lot of evidence that insulin resistance is the central link. Recent studies have also found that intestinal insulin resistance is also involved. Animal models of diabetes and population studies have found that abnormal expression of certain genes associated with lipid metabolism further leads to insulin resistance. The occurrence of atherosclerosis in diabetic patients is associated with a variety of factors, but abnormalities in plasma lipid levels are the most important factors. Studies have shown that the incidence and mortality of cardiovascular disease in diabetic patients is significantly higher than non-diabetic patients, diabetes has become an independent risk factor for cardiovascular disease ^[3] .

In recent years, the relationship between nephropathy and lipid metabolism disorders has become more and more conspicuous. In chronic progressive renal injury, abnormal lipid metabolism is often accompanied, and hyperlipidemia can promote and aggravate kidney damage, in addition to mediating glomerular injury. In addition, it also plays a role in tubulointerstitial damage. In 1913 Munk first described dyslipidemia in nephrotic syndrome. Some scholars have reported that 70%-10% of patients with nephrotic syndrome may have hyperlipidemia. It is mainly characterized by a significant increase in total cholesterol (TC) and a decrease in low-density lipoprotein cholesterol, a slight increase in triglyceride (TG), and an increase in low-density lipoprotein (LDL) and urine protein. There is a certain correlation ^[4] . Patients with chronic renal insufficiency are mainly moderate triglycerideemia, plasma total cholesterol levels are generally normal, and cholesterol is increased in VLDLC and medium density lipoprotein cholesterol (IDLC). High-density lipoprotein cholesterol (HDLC) is reduced, and triglyceride levels are increased in various lipoproteins. The root cause is the adverse effects of uremia environment on the synthesis and catabolism of triglycerides and the inhibition of reverse cholesterol transport ^[5] .

With the widespread use of kidney transplantation therapy and the widespread use of various new immunosuppressive agents (especially CsA, prednisone), the survival of patients with chronic renal failure (CRF) is significantly prolonged, but hyperlipidemia after renal transplantation The incidence is very high. Hyperlipidemia after renal transplantation mainly for plasma total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDLC), very low density lipoprotein cholesterol (VLDLC) levels increased ^[6].

Clinical studies have confirmed a certain correlation between lipid metabolism disorders and diabetic nephropathy. Diabetes patients with lipid metabolism disorders, elevated lipid deposition in the glomerular basement membrane, stimulate basement membrane cell proliferation and extracellular matrix formation. As early as 1936, Kimmelstiel and Wilson found large amounts of lipid deposits in the renal arteries, glomeruli, and renal tubules of patients with diabetic nephropathy ^[7] . Abnormal lipid metabolism makes glomerular and tubulointerstitial fibrosis an important cause of progressive damage to renal function ^[8] .

Disorders of lipid metabolism can also lead to the development of obesity (obesity syndrome). Obesity is divided into two categories: simple and secondary. Simple obesity refers to obesity without obvious endocrine and metabolic diseases. It can be divided into two types: physical obesity and acquired obesity. There is a family history of constitutional obesity. The patient is rich in food intake from childhood, and the amount of excess is obese. The fat cells are hypertrophic and hypertrophic. Acquired obesity is mostly caused by overnutrition and/or reduced physical activity, such as improvement of living conditions after a middle age, adequate recovery of disease and rest, postpartum physical exercise or physical labor. The fat cells showed hypertrophic changes, no hyperplasia, and the treatment effect was better. Secondary obesity is mainly caused by neuroendocrine diseases. Neuroendocrine plays an important role in the regulation of metabolism: 1 The hypothalamus has a center of appetite regulation, central nervous system inflammation sequelae, trauma, tumors, etc. can cause hypothalamic dysfunction, causing an appetite and causing obesity. 2 increased insulin secretion, such as early non-insulin-dependent diabetes mellitus injection of excessive insulin, resulting in hyperinsulinemia; islet B cell tumors secrete too much insulin, which increases fat synthesis, causing obesity. 3 pituitary function is reduced, especially when gonadotropin and thyroid stimulating hormone are reduced, causing obesity and hypothyroidism, obesity can occur. 4 maternal or oral female contraceptives are prone to obesity, suggesting that estrogen has a role in promoting fat synthesis. 5 Cortisol is often accompanied by central obesity. 6 hypothyroidism, due to low metabolic rate, fat accumulation, and mucus edema. 7 hypogonads can also be obese, such as obesity reproductive incompetence (cerebral obesity, Florik's syndrome, trauma, encephalitis, pituitary tumor, craniopharyngioma, etc. caused by damage to the hypothalamus, manifested as central obesity , with diabetes insipidus and sexual growth retardation).

Lipid metabolism disorders often lead to fatty liver. Fatty liver refers to a lesion of excessive accumulation of fat in liver cells due to various reasons. The liver plays a particularly important role in lipid metabolism. It can synthesize lipoproteins, which is beneficial to lipid transport and is also the main site for fatty acid oxidation and ketone body formation. The normal liver fat content is not much, about 4%, mainly phospholipids. If the liver cannot transport the fat out in time, the fat accumulates in the liver cells, which forms fatty liver.

Fatty liver can be an independent disease or can be caused by other causes, such as obesity fatty liver, alcoholic fatty liver, rapid weight loss fatty liver, malnutrition fatty liver, diabetic fatty liver, drug-induced fatty liver and so on.

Certain drugs or chemical poisons cause fatty livers by inhibiting the synthesis of proteins such as tetracycline, adrenocortical hormone, puromycin, cyclohexylamine, ipecaine, and arsenic, lead, silver, mercury, and the like. Lipid lowering drugs can also form fatty liver by interfering with the metabolism of lipoproteins.

One of the dangers of fatty liver is that it promotes the formation of atherosclerosis. One of the causes of atherosclerosis is that patients with fatty liver are often accompanied by hyperlipidemia, increased blood viscosity, and low-density lipoprotein (LDL). Because of its extremely small molecular weight, it easily passes through the intima of the arteries and settles on the vessel wall, which reduces the elasticity of the artery, narrows the diameter of the tube, and weakens the flexibility, eventually leading to blood circulation disorder. The second risk of fatty liver is to induce or aggravate high blood pressure, coronary heart disease, and easily lead to myocardial infarction and sudden death. The third risk of fatty liver is encephalopathy fatty liver syndrome (Reye syndrome). The fourth risk of fatty liver is cirrhosis, liver failure, and liver cancer.

Fatty liver is a product of liver lipid metabolism disorder, and at the same time it is a causative factor that aggravates liver damage. This is a development of mutual causal and vicious circle. The increase of lipid droplets in the liver cells causes the liver cells to be steatotic and swollen, and the nucleus is squeezed off-center. The metabolism of fat should be carried out in the mitochondria. The transport of fat to the extracellular cells mainly passes through the smooth endoplasmic reticulum. The accumulation of fat in the hepatocytes further aggravates the burden of mitochondria and endoplasmic reticulum and reduces its function, thereby affecting other nutrients and hormones. Metabolism of vitamins. Long-term degeneration of hepatocytes leads to arrhythmia and necrosis of hepatocytes, which in turn leads to liver fibrosis and cirrhosis. Cirrhosis is associated with a higher incidence of hepatocellular carcinoma.

The fifth risk of fatty liver is acute pregnancy fatty liver with high mortality. This disease, also known as obstetric acute yellow liver atrophy, is a rare pregnancy with a prognosis. Most occur in the last three months of pregnancy, clinical manifestations are often similar to acute liver, acute liver failure, pancreatitis, renal failure, systemic coagulation abnormalities leading to rapid death, the majority of pregnant women in the first pregnancy.

The sixth risk of fatty liver is to induce or aggravate diabetes. In patients with obese fatty liver, if the blood glucose concentration exceeds the normal level, although it does not meet the diagnostic criteria for diabetes, it is generally considered to be pre-diabetes. Fatty liver and diabetes often accompany each other and affect each other, which brings greater difficulties to clinical treatment.

The present inventors have found that plasminogen can prevent and/or reduce abnormal deposition of fat in tissues and organs of the body, for example, can prevent and reduce abnormal deposition of lipids in tissues of blood, blood vessel walls, internal organs and organs, and improve these. The function of tissues and organs, thus providing a new preventive and therapeutic solution for disorders of fat metabolism and related disorders, as well as its accompanying diseases or complications

Summary of invention

The invention relates to the following items:

In one aspect, the invention relates to: 1. A method of preventing atherosclerosis comprising administering a prophylactically effective amount of plasminogen to a subject susceptible to atherosclerosis.

Item 2. The method of Item 1, wherein the subject susceptible to atherosclerosis is susceptible or suffering from a disorder of fat metabolism, a glucose metabolism disease, a liver disease, a kidney disease, a cardiovascular disease, an intestinal disease, Thyroid disease, gallbladder or biliary tract disease, subjects with obesity, or subjects who drink excessively.

Item 3. The use of Item 2, wherein the subject susceptible to atherosclerosis is susceptible or suffering from hypertension, diabetes, chronic hepatitis, cirrhosis, kidney injury, chronic glomerulonephritis, chronic renal pelvis Nephritis, nephrotic syndrome, renal insufficiency, kidney transplantation, uremia, hypothyroidism, obstructive cholecystitis, obstructive cholangitis.

The method of item 1, wherein the subject susceptible to atherosclerosis is a subject having congenital or secondary obesity.

The method of any one of clauses 1 to 4, wherein the subject susceptible to atherosclerosis is susceptible or suffering from hypertension, diabetes, hyperlipidemia, hyperlipoproteinemia or fat Liver subjects.

In another aspect, the invention relates to: 6. A method of preventing and/or reducing abnormal deposition of fat in a body tissue of a subject comprising administering to the subject an effective amount of plasminogen.

Item 7. The method of Item 6, wherein the abnormal deposition of fat in body tissues and organs refers to abnormal deposition of fat in blood, subcutaneous tissue, blood vessel wall, and internal organs.

In another aspect, the invention relates to: 8. A method of preventing or treating a disease in a subject by attenuating the abnormal deposition of fat in body tissues and organs, comprising administering to the subject an effective amount of plasminogen.

The method of Item 8, wherein the disease comprises obesity, fatty liver, cirrhosis, atherosclerosis, coronary heart disease.

In another aspect, the invention relates to: 10. A method of preventing atherosclerosis in a subject with hyperlipidemia comprising administering to the subject an effective amount of plasminogen.

Item 11. The method of Item 10, wherein the subject has one or more of the following blood lipids: elevated serum total cholesterol levels, elevated serum triglyceride levels, and elevated serum low density lipoprotein levels.

The method of clause 10 or 11, wherein the plasminogen prevents atherosclerosis by one or more of the following: lowering serum total cholesterol levels in the subject, lowering serum triglyceride levels in the subject, decreasing The subject has serum low-density lipoprotein levels and elevated serum high-density lipoprotein levels in the subject.

In another aspect, the invention relates to: 13. A method of preventing atherosclerosis comprising administering a plasminogen in a subject to prevent atherosclerosis by one or more selected from the group consisting of: Promotes fat metabolism in the liver, promotes fat transport in the liver, and reduces fat deposition in the liver of the subject.

Item 14. The method of Item 13, which further comprises preventing atherosclerosis by one or more of the following: lowering serum total cholesterol levels in the subject, lowering serum triglyceride levels in the subject, and lowering serum low density lipid in the subject Protein levels increase serum high-density lipoprotein levels in subjects.

Item 15. The method of Item 13 or 14, further comprising preventing atherosclerosis by reducing deposition of fat in the vessel wall.

In another aspect, the invention relates to: 16. A method of preventing coronary atherosclerosis and/or coronary heart disease in a subject comprising administering to the subject an effective amount of plasminogen.

Item 17. The method of Item 16, wherein the plasminogen prevents coronary atherosclerosis and/or coronary heart disease in a subject by one or more selected from the group consisting of promoting liver fat metabolism and promoting liver fat transport. And reducing fat deposition in the liver of the subject.

Item 18. The method of Item 16 or 17, wherein the plasminogen prevents coronary atherosclerosis and/or coronary heart disease in a subject by one or more selected from the group consisting of: lowering serum total cholesterol levels in the subject, and decreasing The subject has serum triglyceride levels, lowers serum low density lipoprotein levels in the subject, and increases serum high density lipoprotein levels in the subject.

The method of any one of clauses 16-18, wherein the plasminogen prevents coronary atherosclerosis and/or coronary heart disease in a subject by decomposing deposition of fat in the aortic and/or coronary vessel wall.

Item 20. A method of preventing an atherosclerosis-related disorder comprising administering an effective amount of plasminogen to a subject susceptible to atherosclerosis.

Item 21. The method of Item 20, wherein the related condition is a condition caused by arterial stenosis or arterial thrombosis due to atherosclerosis.

The method of item 21, wherein the condition is one or more selected from the group consisting of coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, heart failure, cerebral ischemia, cerebral thrombosis, brain atrophy, cerebral hemorrhage, Cerebral embolism, renal insufficiency, hypertension, glomerular fibrosis, renal failure, uremia, intestinal necrosis, blood in the stool, intermittent claudication, gangrene.

In another aspect, the invention relates to: Item 23. A method of treating hyperlipidemia comprising administering to a subject an effective amount of plasminogen.

The method of Item 23, wherein the hyperlipidemia is one or more selected from the group consisting of elevated serum total cholesterol levels, elevated serum triglyceride levels, and elevated serum low density lipoprotein levels.

In another aspect, the invention relates to: 25. A method of preventing atherosclerosis in a diabetic patient comprising administering an effective amount of plasminogen in a diabetic patient.

In another aspect, the invention relates to: 26. A method of preventing fatty liver comprising administering to a subject an effective amount of plasminogen.

The use according to any one of items 1 to 26, wherein the plasminogen is administered in combination with one or more other drugs and methods of treatment.

The method of item 27, wherein the one or more other drugs include: a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, and a drug Liver drugs, antiarrhythmic drugs, cardiotonic drugs, diuretic drugs, anti-infective drugs, antiviral drugs, immunomodulatory drugs, inflammatory regulating drugs, hormone drugs.

The method of any one of clauses 1 to 28, wherein the plasminogen and the sequence 2, 6, 8, 10 or 12 have at least 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98% or 99% sequence identity and still have plasminogen activity.

The method of any one of clauses 1 to 29, wherein the plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1- 5, 1-4, 1-3, 1-2, 1 amino acid, and still have plasminogen activity protein.

The method of any one of clauses 1 to 30, wherein the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.

The method of any one of items 1 to 31, wherein the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, and delta-fiber Lysogens or their variants that retain plasminogen activity.

The method of any one of items 1 to 3, wherein the plasminogen is a natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.

The method of any one of items 1 to 3, wherein the plasminogen is a human plasminogen ortholog from a primate or a rodent or a plasminogen-retaining activity thereof Body or fragment.

The method of any one of items 1 to 34, wherein the amino acid of the plasminogen is as shown in sequence 2, 6, 8, 10 or 12.

The method of any one of items 1 to 35, wherein the plasminogen is human natural plasminogen.

The method of any one of clauses 1 to 36, wherein the subject is a human.

The method of any one of clauses 1 to 3, wherein the subject lacks or deletes plasminogen.

The method of item 38, wherein the deficiency or deletion is innate, secondary, and/or local.

In another aspect, the invention relates to: Item 40. A plasminogen for use in the method of any of items 1-39.

In another aspect, the invention relates to: 41. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the method of any of items 1-39.

In another aspect, the invention relates to: Item 42. A prophylactic or therapeutic kit comprising: (i) plasminogen for use in the method of any of items 1-39 and (ii) Means for delivering the plasminogen to the subject.

The kit of item 42, wherein the member is a syringe or vial.

Item 42. The kit of Item 42 or 43, further comprising a label or instructions for use, the label or instructions for use instructing administration of the plasminogen to the subject to perform any of items 1-39 Method.

In another aspect, the invention relates to: Item 45. An article comprising:

a container containing a label; and

A pharmaceutical composition comprising (i) plasminogen or a plasminogen-containing composition for use in the method of any one of clauses 1 to 39, wherein the label indicates administration of the plasminogen or composition The subject is the method of any of items 1-39.

The kit of claim 46, or the article of item 45, further comprising one or more additional members or containers containing other drugs.

The kit or article of item 46, wherein the other drug is selected from the group consisting of a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, Hepatoprotective drugs, antiarrhythmic drugs, cardiotonic drugs, diuretic drugs, anti-infective drugs, antiviral drugs, immunomodulatory drugs, inflammatory regulating drugs, anti-tumor drugs, hormone drugs, thyroxine.

The invention also relates to the use of plasminogen for the method of any of items 1-39.

The invention further relates to the use of plasminogen for the preparation of a medicament, a pharmaceutical composition, an article, a kit for use in the method of any of items 1-39.

The invention also relates to the prevention and/or treatment of disorders of fat metabolism and related disorders in a subject.

In one aspect, the invention relates to a method of preventing and/or treating a disorder of fat metabolism and a related disorder thereof, comprising administering to a subject a prophylactically and/or therapeutically effective amount of plasminogen, wherein said subject is susceptible Suffering from fat metabolism disorder, suffering from fat metabolism disorder or suffering from other diseases accompanied by disorders of fat metabolism. The invention also relates to the use of plasminogen for the prevention and/or treatment of disorders of fat metabolism and related disorders in a subject. The invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for the prevention and/or treatment of a disorder of fat metabolism and a related disorder in a subject. Further, the present invention relates to plasminogen for preventing and/or treating a disorder of fat metabolism and a related condition thereof in a subject. The present invention also relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for use in preventing and/or treating a disorder of fat metabolism and a related condition thereof.

In some embodiments, the disorder of fat metabolism is an endocrine disorder disease, a glucose metabolism disease, a liver disease, a kidney disease, a cardiovascular disease, an intestinal disease, a thyroid disease, a gallbladder or a biliary tract disease, an obesity, a drinking, or a medical treatment. Or accompanying disorders of fat metabolism. In some embodiments, the disorder of fat metabolism is hypertension, diabetes, chronic hepatitis, cirrhosis, kidney damage, chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, kidney transplantation, uremia, A disorder of fat metabolism caused by or associated with hypothyroidism, obstructive cholecystitis, obstructive cholangitis, drug or hormonal therapy. In some embodiments, the disorder of fat metabolism is hyperlipidemia, hyperlipoproteinemia, fatty liver, atherosclerosis, obesity, organ fat deposition. In still other embodiments, the atherosclerosis comprises aortic atherosclerosis Hardening, coronary atherosclerosis, cerebral atherosclerosis, renal atherosclerosis, hepatic atherosclerosis, mesenteric atherosclerosis, lower extremity atherosclerosis.

In yet another aspect, the present invention relates to a method of preventing and/or reducing abnormal deposition of fat in a body tissue of a subject, comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for preventing and/or reducing abnormal deposition of fat in a body tissue organ of a subject. The invention further relates to the use of plasminogen for the preparation of a medicament, a pharmaceutical composition, an article, a kit for preventing and/or reducing abnormal deposition of fat in a body tissue of a subject. Further, the present invention relates to plasminogen for preventing and/or reducing abnormal deposition of fat in a body tissue of a subject. The present invention also relates to a medicament, a pharmaceutical composition, an article, a kit comprising plasminogen for preventing and/or reducing abnormal deposition of fat in a body tissue of a subject.

In yet another aspect, the present invention relates to a method of preventing and/or treating a condition caused by abnormal deposition of fat in a body tissue organ of a subject, comprising administering to the subject an effective amount of plasminogen. The invention further relates to the use of plasminogen for preventing and/or treating a condition caused by abnormal deposition of fat in a body tissue or organ of a subject. The invention further relates to the use of plasminogen for the preparation of a medicament, a pharmaceutical composition, an article, a kit for the prevention and/or treatment of a condition in which fat in a subject is abnormally deposited in tissues and organs of the body. Further, the present invention relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for preventing and/or treating a condition caused by abnormal deposition of fat in a body tissue of a subject.

In some embodiments, the abnormal deposition of the fat in the body tissue refers to the abnormal deposition of fat in the blood, subcutaneous tissue, blood vessel walls, internal organs. In some embodiments, the disorder caused by abnormal deposition of fat in body tissues and organs includes obesity, hyperlipidemia, hyperlipoproteinemia, fatty liver, atherosclerosis, lipid heart damage, lipidity Kidney damage, lipid islet damage.

In still another aspect, the present invention relates to a method of preventing and/or treating a condition caused by a disorder of fat metabolism in a subject, comprising administering to the subject an effective amount of plasminogen. The invention further relates to the use of plasminogen for the prevention and/or treatment of a condition caused by a disorder of fat metabolism in a subject. The invention further relates to the use of plasminogen for the preparation of a medicament, a pharmaceutical composition, an article, a kit for the prevention and/or treatment of a disorder caused by a disorder of fat metabolism in a subject. Further, the present invention relates to plasminogen for preventing and/or treating a disorder caused by a disorder of fat metabolism in a subject. The present invention also relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for preventing and/or treating a disorder caused by a disorder of fat metabolism in a subject. In some embodiments, the condition comprises obesity, hyperlipidemia, high Lipoproteinemia, fatty liver, atherosclerosis, lipid heart tissue damage, lipid-induced kidney injury.

In yet another aspect, the present invention relates to a method of treating a disease in a subject by reducing fat abnormal deposition comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for treating diseases in a subject by reducing fat abnormal deposition. The invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for treating a disease in a subject by reducing fat abnormal deposition. Further, the present invention also relates to plasminogen for treating a disease in a subject by reducing fat abnormal deposition. The present invention also relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for treating a disease in a subject by reducing fat abnormal deposition.

In some embodiments, the disease comprises atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, fatty liver, cirrhosis, cerebral ischemia, cerebral infarction, renal insufficiency, nephrotic syndrome, renal insufficiency Obesity.

In yet another aspect, the present invention relates to a method of preventing and/or treating lipid damage in a tissue of a subject comprising administering to the subject an effective amount of plasminogen. The invention further relates to the use of plasminogen for the prevention and/or treatment of lipid damage in tissues and organs of a subject. The invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for the prevention and/or treatment of lipid damage in tissues and organs of a subject. Further, the present invention relates to plasminogen for preventing and/or treating lipid damage in tissues and organs of a subject. The present invention also relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for preventing and/or treating lipid damage in tissues and organs of a subject.

In some embodiments, the tissue organ comprises an arterial wall, a heart, a liver, a kidney, a pancreas.

In yet another aspect, the invention relates to a method of improving hyperlipidemia in a subject comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for improving hyperlipidemia in a subject. The invention further relates to the use of plasminogen for the preparation of a medicament, a pharmaceutical composition, an article, a kit for improving hyperlipidemia in a subject. Further, the present invention also relates to plasminogen for improving hyperlipidemia in a subject. The present invention also relates to a medicament, a pharmaceutical composition, an article, a kit comprising plasminogen for improving hyperlipidemia in a subject.

In some embodiments, the hyperlipidemia is selected from one or more of the group consisting of hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia, and low high density lipoproteinemia.

In yet another aspect, the invention relates to a method of reducing the risk of atherosclerosis in a subject comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for reducing the risk of atherosclerosis in a subject. The invention further relates to the use of plasminogen in the manufacture of a medicament, pharmaceutical composition, article, kit for reducing the risk of atherosclerosis in a subject. Further, the present invention also relates to plasminogen for reducing the risk of atherosclerosis in a subject. The invention also relates to a medicament, pharmaceutical composition, preparation, kit comprising plasminogen for reducing the risk of atherosclerosis in a subject.

In some embodiments, the subject has hypertension, obesity, diabetes, chronic hepatitis, cirrhosis, kidney injury, chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, kidney transplantation , uremia, hypothyroidism, obstructive cholecystitis or obstructive cholangitis, or the subject taking a drug or hormone that affects fat metabolism. In some embodiments, the plasminogen reduces the risk of atherosclerosis in a subject by one or more of the following: lowering total cholesterol levels in the blood, triglyceride levels, low density lipoprotein levels, Increase blood HDL levels.

In yet another aspect, the invention relates to a method of treating a disease by ameliorating hyperlipidemia in a subject comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for the treatment of diseases by improving hyperlipidemia in a subject. The invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for the treatment of a disease by improving hyperlipidemia in a subject. Further, the present invention also relates to plasminogen for treating diseases by improving hyperlipidemia in a subject. The present invention also relates to a medicament, a pharmaceutical composition, an article, a kit comprising plasminogen for treating a disease by improving hyperlipidemia in a subject.

In some embodiments, the condition comprises diabetes, hypertension, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, chronic hepatitis, fatty liver, cirrhosis, cerebral insufficiency, cerebral ischemia, cerebral infarction Chronic nephritis, chronic pyelonephritis, renal insufficiency, nephrotic syndrome, uremia, obesity.

In yet another aspect, the present invention relates to a method of preventing and/or treating a hyperlipidemia-related disorder in a subject comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for the prevention and/or treatment of a hyperlipidemia-related disorder in a subject. The invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for the prevention and/or treatment of a hyperlipidemic disorder in a subject. Further, the present invention also relates to plasminogen for preventing and/or treating a hyperlipidemia-related disorder in a subject. The invention also relates to a method for preventing and/or treating hyperlipidemia associated with a subject Drug, pharmaceutical composition, preparation, and kit containing plasminogen. In some embodiments, the condition comprises diabetes, hypertension, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, chronic hepatitis, fatty liver, cirrhosis, cerebral insufficiency, cerebral ischemia, cerebral infarction Chronic nephritis, chronic pyelonephritis, renal insufficiency, nephrotic syndrome, uremia, obesity.

In any of the above embodiments of the invention, the plasminogen can be administered in combination with one or more other drugs or methods of treatment. In some embodiments, the one or more other drugs include a therapeutic drug for hypertension, a drug for treating diabetes, a drug for treating atherosclerosis, a drug for treating chronic glomerulonephritis, a drug for treating chronic pyelonephritis, and a combination of nephropathy Drugs for treatment, drugs for treatment of renal insufficiency, drugs for uremia treatment, drugs for kidney transplantation, drugs for treatment of fatty liver, drugs for treatment of liver cirrhosis, drugs for treatment of obesity. In some embodiments, the other drug comprises: a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a hepatoprotective drug, an antiarrhythmic drug, Cardiotonic drugs, diuretic drugs, anti-infective drugs, antiviral drugs, immunomodulatory drugs, inflammatory regulating drugs, anti-tumor drugs, hormone drugs, thyroxine. In some further embodiments, the medicament comprises a hypolipidemic drug: a statin; a fibrate; a niacin; a cholestyramine; clofibrate; an unsaturated fatty acid such as erosin, a blood lipid, and a heart pulse; alginic acid Sodium diester; antiplatelet drugs: aspirin; dipyridamole; clopidogrel; cilostazol; dilated vascular drugs: hydralazine; nitroglycerin and heartache; sodium nitroprusside; alpha nitrate receptor blockers such as prazosin ; alpha receptor blockers such as phentolamine; beta pull receptor stimulants such as salbutamol; captopril, enalapril; heart pain, thiazolone; lysine, long pressure , prostaglandins, atrial natriuretic peptide; thrombolytic drugs: urokinase and streptokinase; tissue plasminogen activator; single-chain urokinase-type plasminogen activator; TNK-tissue plasminogen activator; Anticoagulant drugs: heparin; enoxaparin; nadroparin; bivalirudin.

In any of the above embodiments of the invention, the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97 with sequence 2, 6, 8, 10 or 12. %, 98% or 99% sequence identity and still have plasminogen activity. In some embodiments, the plasminogen is added, deleted, and/or substituted on the basis of sequence 2, 6, 8, 10, or 12, 1-100, 1-90, 1-80, 1-70 , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid, and still a protein with plasminogen activity.

In some embodiments, the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity. In some embodiments, the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta-plasminogen, or their retention A variant of plasminogen activity. In some embodiments, the plasminogen is a native or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or a rodent or a variant or fragment thereof that still retains plasminogen activity. In some embodiments, the amino acid of plasminogen is as shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is human natural plasminogen.

In some embodiments, the subject is a human. In some embodiments, the subject lacks or lacks plasminogen. In some embodiments, the deficiency or deficiency is innate, secondary, and/or local.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and plasminogen for use in the foregoing methods. In some embodiments, the kit can be a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the foregoing methods and (ii) for delivery of the plasminogen to The subject of the subject. In some embodiments, the member is a syringe or vial. In some embodiments, the kit further comprises a label or instructions for use, the label or instructions for use indicating administration of the plasminogen to the subject to perform any of the methods described above.

In some embodiments, the article of manufacture comprises: a container comprising a label; and a pharmaceutical composition comprising (i) plasminogen or a plasminogen for use in the foregoing method, wherein the label indicates that the fiber is to be The lysogen or composition is administered to the subject to perform any of the methods described above.

In some embodiments, the kit or article further comprises an additional one or more components or containers containing other drugs. In some embodiments, the other drug is selected from the group consisting of a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a hepatoprotective drug, and an anti-heart rhythm Abnormal drugs, cardiotonic drugs, diuretic drugs, anti-infective drugs, antiviral drugs, immunomodulatory drugs, inflammatory regulatory drugs, anti-tumor drugs, hormone drugs, thyroxine.

In some embodiments of the foregoing methods, the plasminogen is administered systemically or locally, preferably by the following route: intravenous, intramuscular, subcutaneous administration of plasminogen for treatment. In some embodiments of the aforementioned methods, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In some embodiments of the aforementioned methods, the plasminogen is 0.0001 per day 2000mg/kg, 0.001-800mg/kg, 0.01-600mg/kg, 0.1-400mg/kg, 1-200mg/kg, 1-100mg/kg, 10-100mg/kg (calculated per kg body weight) or 0.0001-2000mg Dosage administration of /cm2, 0.001-800 mg/cm2, 0.01-600 mg/cm2, 0.1-400 mg/cm2, 1-200 mg/cm2, 1-100 mg/cm2, 10-100 mg/cm2 (calculated as body surface area per square centimeter) Preferably, it is repeated at least once, preferably at least daily.

The present invention expressly covers all combinations of the technical features between the embodiments of the present invention, and these combined technical solutions are explicitly disclosed in the present application, just as the above technical solutions have been separately and explicitly disclosed. In addition, the present invention also explicitly covers combinations between various embodiments and elements thereof, and the combined technical solutions are explicitly disclosed herein.

definition

The "fat metabolism disorder" according to the present invention is also called "abnormal fat metabolism" and "fat metabolism disorder", and is a general term for clinical or pathological manifestations caused by abnormal, disorder or disorder of fat metabolism. Herein, "fat metabolism disorder", "abnormal fat metabolism", and "fat metabolic disorder" are used interchangeably. In the present invention, "fat metabolism", "lipid metabolism", and "lipid metabolism" are used interchangeably.

"Fat metabolism disorder-related disorders" is a general term for disorders associated with disorders of fat metabolism. The correlation may be related to etiology, pathogenesis, pathological relevance, clinical symptom correlation, and/or treatment principles.

"Blood fat" is a general term for triglycerides, cholesterol and phospholipids. Lipoprotein is a spherical macromolecular complex composed of apolipoprotein and blood lipid. Because lipoproteins contain different components of cholesterol and triglycerides, and the density is different. Divided into five categories: chylomicrons (CM) very low density lipoprotein (VLDL) medium density lipoprotein (IDL) low density lipoprotein (LDL) high density lipoprotein (HDL). According to the level of lipid risk, the most common types of dyslipoproteinemia in clinical practice: hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia, and low-density lipoproteinemia. Secondary dyslipidemia is found in diabetes, hypothyroidism, nephrotic syndrome, kidney transplantation, severe liver disease, obstructive biliary tract disease, obesity, alcohol consumption, drug treatment, such as estrogen therapy, etc., if secondary dyslipidemia can be ruled out Consider primary dyslipidemia.

"Hyperlipidemia" refers to a pathological condition in which blood lipids such as cholesterol, triglyceride, phospholipids, and non-lipidated fatty acids are increased in plasma.

"Hyperlipid-related disorder" refers to a condition associated with high blood lipids, etiology, pathogenesis, pathological manifestations, clinical symptoms, and/or treatment principles. Preferably the condition includes, but is not limited to, diabetes, high blood Compression, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, chronic hepatitis, fatty liver, cirrhosis, cerebral insufficiency, cerebral ischemia, cerebral infarction, chronic nephritis, chronic pyelonephritis, renal insufficiency, nephropathy Syndrome, uremia, obesity.

One or several lipid abnormalities in plasma are referred to as "hyperlipidemia", "hyperlipemia" or "dyslipidemia" due to fat metabolism or abnormal function.

Because lipids are insoluble or slightly soluble in water, they must bind to proteins to form lipoproteins to function in the blood circulation. Therefore, hyperlipidemia is often a reflection of "high-lipoproteinemia."

The "hyperlipidemia-related disorder" of the present invention may also be referred to as "hyperlipidemic-related disorder" and "hyperlipoproteinemia-related disorder".

"Fatty liver" refers to a lesion of excessive accumulation of fat in liver cells due to various reasons, which may be an independent disease or may be caused by other causes, such as obese fatty liver, alcoholic fatty liver, and rapid weight loss. Fatty liver, malnutrition fatty liver, diabetic fatty liver, drug-induced fatty liver and so on.

In the case of fatty liver, the lipid droplets in the liver cells increase, causing the liver cells to be fatty degenerated, swollen, and the nucleus is squeezed off center. The metabolism of fat should be carried out in the mitochondria. The transport of fat to the extracellular cells mainly passes through the smooth endoplasmic reticulum. The accumulation of fat in the hepatocytes further aggravates the burden of mitochondria and endoplasmic reticulum and reduces its function, thereby affecting other nutrients and hormones. Metabolism of vitamins. Long-term degeneration of hepatocytes leads to arrhythmia and necrosis of hepatocytes, which in turn leads to liver fibrosis and cirrhosis.

"Atherosclerosis" is a chronic, progressive arterial disease in which the fat deposited in the arteries partially or completely blocks blood flow. Atherosclerosis occurs when the smooth, firm arterial intima is roughened, thickened, and blocked by fat, fibrin, calcium, and cellular debris. Atherosclerosis is a gradual process. When the concentration of lipids in the blood is greatly increased, fat streaks are formed along the walls of the artery. These streaks cause fat and cholesterol deposits that attach to the originally smooth arterial intima, forming a nodule. These nodules then grow fibrotic scar tissue, resulting in calcium deposition. The deposited calcium gradually evolves into a chalky hard film (called an atheroma) that cannot be removed. This permanent film inside the artery blocks the normal expansion and contraction of the artery, slowing the blood flow velocity within the artery, which can easily form a blood clot that prevents or prevents blood from flowing through the artery.

The exact cause of atherosclerosis has not been determined, but important pathogenic factors have been identified: hyperlipidemia, hypertension, history of smoking, and a family history of atherosclerosis (60 years old) Have had the disease before) or diabetes. Hyperlipidemia can promote the formation of fat streaks. Because hypertension exerts a certain constant force on the artery, it accelerates the process of arterial occlusion and hardening, thus increasing the prevalence of atherosclerosis. Smoking can cause arterial contractions and restrict blood flow, thus creating conditions for arterial occlusion. Diabetes can also contribute to the development of atherosclerosis, especially for very small arteries.

As far as atherosclerosis is concerned, people do not feel any symptoms. The disease is only discovered when the artery connected to an important organ in the body is blocked. Symptoms caused by blocked arteries in the organ are more pronounced. For example, if the heart's blood supply artery is partially blocked, people may feel angina; but if it is completely blocked, it may lead to heart disease (heart tissue death from the blocked arteries). If atherosclerosis affects the brain arteries, people may feel dizziness, blurred vision and syncope, and may even cause a stroke (the brain tissue that is supplied by the blocked arteries dies, causing nerve damage, such as limbs controlled by dead brain tissue) ). Blockage of the arteries leading to the kidney may also lead to kidney failure. Blockage of blood vessels leading to the eye can lead to blindness. Arterial obstruction of the extremities may cause lesions in various limbs.

Atherosclerosis is the main cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. Lipid metabolism disorder is the basis of atherosclerosis, which is characterized by the involvement of the affected arterial lesions from the intima, usually the accumulation of lipids and complex carbohydrates, hemorrhage and thrombosis, and then fibrous tissue hyperplasia and calcium deposition, and The gradual metamorphosis and calcification of the middle layer of the artery leads to thickening and hardening of the arterial wall and narrowing of the vascular lumen. The lesion often involves the large and medium muscular arteries, and once developed enough to block the arterial lumen, the tissue or organ supplied by the artery will be ischemic or necrotic.

Atherosclerosis is a systemic disease in which an organ angiogenic atherosclerotic lesion means that the same lesion may already exist in the blood vessels elsewhere; likewise, a vascular event in one organ means that a vascular event occurs elsewhere. The danger increases.

Detailed description of the invention

Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that hydrolyzes several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans ^[9] . In addition, plasmin activates some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis ^[10,11] . Plasmin is formed by proteolytic plasminogen by two physiological PAs: tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). Due to the relatively high levels of plasminogen in plasma and other body fluids, it has been traditionally believed that the regulation of the PA system is primarily achieved by the synthesis and activity levels of PAs. The synthesis of components of the PA system is tightly regulated by various factors such as hormones, growth factors and cytokines. In addition, specific physiological inhibitors of plasmin and PAs are also present. The main inhibitor of plasmin is α2-antiplasmin. The activity of PAs is simultaneously regulated by plasminogen activator inhibitor-1 (PAI-1), which inhibits uPA and tPA, and lysogen activator inhibitor-2 (PAI-2), which mainly inhibits uPA. Some cell surface has direct hydrolysis activity of uPA-specific cell surface receptors (uPAR) ^[12,13] .

Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa ^[14,15] . Plasminogen is mainly synthesized in the liver and is abundantly present in the extracellular fluid. The plasma plasminogen content is approximately 2 μM. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids ^[16,17] . Plasminogen exists in two molecular forms: glutamate-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved forms of plasminogen have an amino terminal (N-terminal) glutamate and are therefore referred to as glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed to Lys-Lysinogen at Lys76-Lys77. Compared to glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate. The Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide-linked double-chain protease plasmin ^[18] . The amino terminal portion of plasminogen contains five homologous tricycles, the so-called kringles, which contain a protease domain. Some kringles contain a lysine binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor alpha2-AP. A new fragment of plasminogen of 38 kDa, including kringles 1-4, was recently discovered to be a potent inhibitor of angiogenesis. This fragment was named angiostatin and can be produced by hydrolysis of plasminogen by several proteases.

The main substrate for plasmin is fibrin, which is the key to preventing pathological thrombosis ^[19] . Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycans and gelatin, suggesting that plasmin also plays an important role in ECM reconstruction [ ^{15, 20, 21]} . Indirectly, plasmin can also degrade other components of ECM, including MMP-1, MMP-2, MMP-3 and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis ^[22] . In addition, plasmin has the ability to activate certain potential forms of growth factors ^[23-25] . In vitro, plasmin also hydrolyzes components of the complement system and releases chemotactic complement fragments.

"plasmin" is a very important enzyme found in the blood that hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

"plasminogen" is a zymogen form of plasmin, which is composed of 810 amino acids, based on the sequence in swiss prot, based on the native human plasminogen amino acid sequence (sequence 4) containing the signal peptide. 90 kD, a glycoprotein synthesized mainly in the liver and capable of circulating in the blood, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO:3. Full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle 1-5). Referring to the sequence in swiss prot, the signal peptide includes the residue Metl-Gly19, PAp includes the residue Glu20-Val98, Kringle1 includes the residue Cys103-Cys181, Kringle2 includes the residue Glu184-Cys262, and Kringle3 includes the residue Cys275-Cys352, Kringle4 Including the residue Cys377-Cys454, Kringle5 includes the residue Cys481-Cys560. According to NCBI data, the serine protease domain includes the residues Val581-Arg804.

Glu-plasminogen is a natural full-length plasminogen consisting of 791 amino acids (not containing a 19 amino acid signal peptide), and the cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is sequence 2. Shown. In vivo, there is also a Lys-plasminogen which is hydrolyzed from amino acids 76-77 of Glu-plasminogen, and as shown in SEQ ID NO: 6, the cDNA sequence encoding the amino acid sequence is as shown in SEQ ID NO: 5 Shown. Delta-plasminogen is a fragment of full-length plasminogen deleted from the Kringle2-Kringle5 structure and contains only the Kringle1 and serine protease domains ^[26,27] . The delta-plasminogen has been reported in the literature. The amino acid sequence (SEQ ID NO: 8) ^[27] , the cDNA sequence encoding the amino acid sequence is shown in Sequence 7. Mini-plasminogen consists of Kringle5 and a serine protease domain, which has been reported in the literature to include the residue Val443-Asn791 (starting amino acid with a Glu residue of Glu-plasminogen sequence not containing a signal peptide) ^[28] , the amino acid sequence thereof is shown in SEQ ID NO: 10, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9. Micro-plasminogen contains only the serine protease domain, and its amino acid sequence has been reported to include the residue Ala543-Asn791 (from the Glu residue of the Glu-plasminogen sequence containing no signal peptide). Starting from amino acid) ^[29] , there is also a patent document CN102154253A reporting that the sequence includes the residue Lys531-Asn791 (the Glu residue of the Glu-plasminogen sequence not containing the signal peptide is the starting amino acid), and the patent sequence refers to the patent document. CN102154253A, the amino acid sequence of which is shown in SEQ ID NO: 12, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 11.

The "plasmin" of the present invention is used interchangeably with "fibrinolytic enzyme" and "fibrinolytic enzyme", and has the same meaning; "plasminogen" and "plasminogen", "fibrinogenase" "Interchangeable use, meaning the same.

In the present application, the term "deficiency" of plasminogen means that the content or activity of plasminogen in the subject is lower than that of a normal person, and is low enough to affect the normal physiological function of the subject; The meaning of "deficient" of plasminogen is that the content or activity of plasminogen in the subject is significantly lower than that of normal people, and even the activity or expression is minimal, and only by external supply can maintain normal physiological functions.

It will be understood by those skilled in the art that all of the technical solutions of the plasminogen of the present invention are applicable to plasmin. Therefore, the technical solutions described in the present invention encompass plasminogen and plasmin.

During the circulatory process, plasminogen adopts a closed inactive conformation, but when bound to the surface of a thrombus or cell, it is converted to openness mediated by plasminogen activator (PA). Conformational active plasmin. The active plasmin further hydrolyzes the fibrin clot into a fibrin degradation product and a D-dimer, thereby dissolving the thrombus. The PAp domain of plasminogen contains an important determinant that maintains plasminogen in an inactive blocking conformation, while the KR domain is capable of binding to lysine residues present on the receptor and substrate. A variety of enzymes are known that act as plasminogen activators, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hag Mann factor) and so on.

A "plasminogen active fragment" refers to an active fragment that binds to a target sequence in a substrate and exerts a proteolytic function in a plasminogen protein. The technical solution of the present invention relating to plasminogen covers the technical solution of replacing plasminogen with a plasminogen active fragment. The plasminogen active fragment of the present invention is a protein comprising a serine protease domain of plasminogen. Preferably, the plasminogen active fragment of the present invention comprises the sequence 14, and the sequence 14 has at least 80%, 90. A protein of amino acid sequence of %, 95%, 96%, 97%, 98%, 99% homology. Thus, the plasminogen of the present invention comprises a protein comprising the plasminogen active fragment and still retaining the plasminogen activity.

Currently, blood plasminogen and its activity assays include: detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t-PAAg), Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen Detection of activator inhibitor antigen, plasma plasmin-anti-plasmin complex assay (PAP). The most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the plasma to be tested, and the PLG in the tested plasma is converted into PLM under the action of SK, and the latter acts on The chromogenic substrate is then measured spectrophotometrically and the increase in absorbance is directly proportional to the plasminogen activity. In addition, plasminogen activity in blood can also be measured by immunochemical method, gel electrophoresis, immunoturbidimetry, or radioimmunoassay.

"Ortholog or ortholog" refers to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs, orthologs. It specifically refers to a protein or gene that has evolved from the same ancestral gene in different species. The plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs of plasminogen activity derived from different species.

"Conservative substitution variant" refers to a change in one of the given amino acid residues without altering the overall conformation and function of the protein or enzyme, including but not limited to similar properties (eg, acidic, basic, hydrophobic, etc.) The amino acid replaces the amino acid in the amino acid sequence of the parent protein. Amino acids having similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and are interchangeable. Similarly, isoleucine is a hydrophobic amino acid that can be replaced by leucine, methionine or valine. Therefore, the similarity of two protein or amino acid sequences of similar function may be different. For example, based on the similarity (identity) of 70% to 99% of the MEGALIGN algorithm. "Conservative substitution variants" also includes determining polypeptides or enzymes having more than 60% amino acid identity by BLAST or FASTA algorithm. If it is more than 75%, preferably more than 85%, or even more than 90%. Optimal and have the same or substantially similar properties or functions as the native or parent protein or enzyme.

"Isolated" plasminogen refers to a plasminogen protein that is isolated and/or recovered from its natural environment. In some embodiments, the plasminogen will purify (1) to a purity greater than 90%, greater than 95%, or greater than 98% by weight, as determined by the Lowry method, eg, over 99% (by weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is by use Coomassie blue or silver staining was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions. Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymeric form of an amino acid of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived. Amino acids, and polypeptides having a modified peptide backbone. The term includes fusion proteins including, but not limited to, fusion proteins having a heterologous amino acid sequence, fusions having heterologous and homologous leader sequences (with or without an N-terminal methionine residue);

The "percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the introduction of a gap as necessary to achieve maximum percent sequence identity, and without any conservative substitution being considered as part of sequence identity, in the candidate sequence The percentage of amino acid residues that are identical in amino acid residues in the reference polypeptide sequence. Comparisons for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum contrast over the full length of the sequences being compared. However, for the purposes of the present invention, amino acid sequence identity percent values are generated using the sequence comparison computer program ALIGN-2.

In the case where ALIGN-2 is used to compare amino acid sequences, the amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as having or comprising relative to, and, or for a given amino acid sequence) A given amino acid sequence A of a certain % amino acid sequence identity of B is calculated as follows:

Score X/Y times 100

Wherein X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 in the A and B alignments of the program, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. All % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless explicitly stated otherwise.

As used herein, the terms "treating" and "treating" refer to obtaining a desired pharmacological and/or physiological effect. The effect may be to completely or partially prevent the disease or its symptoms, and/or to partially or completely cure the disease and/or its symptoms, and includes: (a) preventing the disease from occurring in the subject, the subject may have The cause of the disease, but not yet diagnosed as having a disease; (b) inhibiting the disease, ie, retarding its formation; and (c) reducing the disease and/or its symptoms, ie causing the disease and/or its symptoms to subside.

The terms "individual", "subject" and "patient" are used interchangeably herein to refer to a mammal, including but not limited to a mouse (rat, mouse), a non-human primate, a human, a dog, a cat. Hoofed animals (such as horses, cattle, sheep, pigs, goats).

"Therapeutically effective amount" or "effective amount" refers to an amount of plasminogen sufficient to effect such prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease. The "therapeutically effective amount" will vary depending on the plasminogen used, the severity of the disease and/or its symptoms of the subject to be treated, and the age, weight, and the like.

Preparation of plasminogen of the invention

Plasminogen can be isolated and purified from nature for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is chemically synthesized, it can be synthesized in a liquid phase or a solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence, is a suitable method for the chemical synthesis of plasminogen. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen. Techniques for solid phase synthesis are described in Barany and Solid-Phase Peptide Synthesis; on page 3-284 in The Peptides: Analysis, Synthesis, Biology. Volume 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. .Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al, Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12: 723-8. Briefly, small insoluble porous beads are treated with functional units on which the peptide chains are constructed. After repeated cycles of coupling/deprotection, the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected to reveal a new N-terminal amine that can be attached to other amino acids. The peptide remains immobilized on the solid phase and then cut off.

The plasminogen of the present invention can be produced using standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector operably linked to a regulatory sequence in an expression vector. Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Expression regulation can be a eukaryotic promoter system in a vector that is capable of transforming or transfecting eukaryotic host cells (eg, COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of plasminogen.

Suitable expression vectors are typically replicated as an episome in the host organism or as an integral part of the host chromosomal DNA. Typically, expression vectors contain a selection marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the desired DNA sequence with foreign sources. Those cells are tested.

Escherichia coli is an example of a prokaryotic host cell that can be used to clone a subject antibody-encoding polynucleotide. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Genus (Pseudomonas) species. In these prokaryotic hosts, expression vectors can also be generated which will typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell. In addition, there are many well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda. Promoters typically control expression, optionally in the context of manipulating a gene sequence, and have a ribosome binding site sequence, etc., to initiate and complete transcription and translation.

Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells in which a suitable vector has expression control sequences (e.g., a promoter), an origin of replication, a termination sequence, and the like, as desired. A typical promoter comprises 3-phosphoglycerate kinase and other saccharolytic enzymes. Inducible yeast is initiated by a promoter specifically comprising an alcohol dehydrogenase, an isocytochrome C, and an enzyme responsible for the utilization of maltose and galactose.

In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell culture) can also be used to express and produce an anti-Tau antibody of the invention (e.g., a polynucleotide encoding a subject anti-Tau antibody). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells may contain expression control sequences such as origins of replication, promoters and enhancers (Queen et al, Immunol. Rev. 89: 49 (1986)), as well as necessary processing information sites, such as ribosome binding. Sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from the white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148: 1149 (1992).

Once synthesized (chemically or recombinantly), the invention may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like. Plasminogen. The plasminogen is substantially pure, such as at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure. Or more pure, for example, free of contaminants, such as cellular debris, macromolecules other than the subject antibody, and the like.

Pharmaceutical formulation

The jelly can be formed by mixing plasminogen of the desired purity with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)). The therapeutic formulation is prepared as a dry formulation or as an aqueous solution. Acceptable carriers, excipients, and stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as Octadecyldimethylbenzylammonium chloride; chlorinated hexane diamine; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptide (less than about 10 residues) Protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharide, Sugars and other carbohydrates include glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, fucose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg zinc-protein) Complex); and/or nonionic surfactants such as TWEENT M, PLURONICSTM or polyethylene glycol (PEG). Preferably, the lyophilized anti-VEGF antibody formulation is described in WO 97/04801, which is incorporated herein by reference.

The formulations of the invention may also contain more than one active compound as required for the particular condition being treated, preferably those having complementary activities and no side effects to each other. For example, antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, and the like.

The plasminogen of the present invention may be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, for example, may be placed in a glial drug delivery system (eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The plasminogen of the invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after lyophilization and reconstitution.

The plasminogen of the present invention can prepare a sustained release preparation. Suitable examples of sustained release formulations include solid hydrophobic polymeric semi-permeable matrices having a shape and containing glycoproteins, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al, J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem .Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S. Patent 3,739,919, EP 58,481), L-glutamic acid and γ-ethyl-L-glutamic acid Copolymer (Sidman, et al, Biopolymers 22: 547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al, supra), or degradable lactic acid-glycolic acid copolymer Such as Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly D-(-)-3-hydroxybutyric acid. Ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, while some hydrogels release proteins for a short time. A reasonable strategy for protein stabilization can be designed according to the relevant mechanism. For example, if agglomeration is found The mechanism is to form intermolecular SS bonds by thiodisulfide bond exchange, which can be modified by thiol residues, lyophilized from acidic solution, and controlled. Degree, using appropriate additives, and developing specific polymer matrix compositions stable.

Dosing and dosage

Administration of the pharmaceutical compositions of this invention may be effected intramuscularly in different ways, such as by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery).

Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffering media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles contain liquid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.

The medical staff will determine the dosage regimen based on various clinical factors. As is well known in the medical arts, the dosage of any patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, number and route of administration, overall health, and other medications administered simultaneously. . The dosage range of the pharmaceutical composition of the invention comprising plasminogen can be, for example, for example Subjects from about 0.0001 to 2000 mg/kg, or from about 0.001 to 500 mg/kg (eg 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 10 mg/kg, 50 mg/kg, etc.) body weight. For example, the dose can be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Dosages above or below this exemplary range are also contemplated, particularly in view of the above factors. Intermediate doses in the above ranges are also included in the scope of the present invention. The subject can administer such doses daily, every other day, every week, or according to any other schedule determined by empirical analysis. An exemplary dosage schedule includes 1-10 mg/kg for several days. The therapeutic effect and safety need to be evaluated in real time during the administration of the drug of the present invention.

Product or kit

One embodiment of the invention relates to an article or kit comprising a plasminogen or plasmin of the invention useful for treating a cardiovascular disease caused by diabetes and a related disorder thereof. The article preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, and the like. The container can be made of various materials such as glass or plastic. The container contains a composition that is effective to treat a disease or condition of the invention and has a sterile access port (eg, the container can be an intravenous solution or vial containing a stopper that can be penetrated by a hypodermic needle) of). At least one active agent in the composition is plasminogen/plasmin. The label on or attached to the container indicates that the composition is used to treat the cardiovascular disease caused by diabetes and its related conditions of the present invention. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes. In addition, the article comprises a package insert with instructions for use, including, for example, a user instructing the composition to administer the plasminogen composition and other drugs to treat the accompanying disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Results of serum atherosclerosis index after 20 days of plasminogen administration in 3% cholesterol hyperlipidemia model mice. The results showed that the atherosclerosis index of the plasminogen group was significantly lower than that of the vehicle PBS control group, and the statistical difference was extremely significant (** means P<0.01). This indicates that plasminogen can effectively reduce the risk of atherosclerosis in mice with hyperlipidemia.

Figure 2 shows the results of serum total cholesterol test after 30 days of administration of plasminogen in ApoE atherosclerotic model mice. The results showed that the total cholesterol concentration in the plasminogen group was significantly lower than that in the vehicle. The PBS control group had statistically significant differences (* indicates P<0.05). This indicates that plasminogen can reduce the total cholesterol in the serum of ApoE atherosclerotic model mice and improve the dyslipidemia in atherosclerotic model mice.

Figure 3 shows the results of serum triglyceride test after 30 days of administration of plasminogen in ApoE atherosclerotic model mice. The results showed that the concentration of triglyceride in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P<0.05). This indicates that plasminogen can reduce the serum triglyceride content of ApoE atherosclerosis model mice and improve dyslipidemia in atherosclerotic model mice.

Figure 4 shows the results of serum low density lipoprotein cholesterol test after 30 days of plasminogen administration in ApoE atherosclerotic model mice. The results showed that the concentration of LDL-C in the plasminogen group was significantly lower than that in the vehicle-treated PBS control group, and the statistical difference was significant (* indicates P<0.05). This indicates that plasminogen can reduce the content of low-density lipoprotein cholesterol in serum of ApoE atherosclerotic model mice and improve dyslipidemia in atherosclerotic model mice.

Figure 5 is a representative picture of liver oil red O staining after 30 days of plasminogen administration in ApoE atherosclerotic model mice. A is the control vehicle PBS control group, B is the plasminogen group, and C is the quantitative analysis result. The results showed that the liver fat deposition in the plasminogen group was significantly less than that in the vehicle control group, and the statistical analysis was statistically significant (* indicates P<0.05). This indicates that plasminogen can reduce the deposition of fat in the liver of atherosclerotic model mice.

Figure 6 is a representative picture of aortic sinus oil red O staining after 30 days of plasminogen administration in ApoE atherosclerotic model mice. A is the vehicle PBS control group and B is the plasminogen group. The results showed that the aortic sinus fat deposition in the plasminogen group was significantly less than that in the vehicle PBS control group. This indicates that plasminogen can improve the deposition of fat in the aortic sinus of atherosclerotic model mice.

Fig. 7 Results of liver oil red O staining after administration of plasminogen for 30 days in a 16-week hyperlipidemia model mouse. A is the control vehicle PBS control group, B is the plasminogen group, and C is the quantitative analysis result. The results showed that the liver fat deposition in the plasminogen group was significantly less than that in the vehicle control group, and the statistical analysis was statistically significant (* indicates P<0.05). This indicates that plasminogen can improve the deposition of fat in the liver of hyperlipidemia model mice.

Figure 8. Results of aortic sinus oil red O staining after administration of plasminogen for 30 days in a 16-week hyperlipidemia model mouse. A and C are the control group for the vehicle PBS, B and D are for the plasminogen group, and E is the quantitative analysis result. The results showed that the aortic sinus fat deposition in the plasminogen group was significantly less than that given The vehicle was in the PBS control group, and the statistical difference was significant (* indicates P<0.05). This indicates that plasminogen can improve the deposition of fat in the aortic sinus of hyperlipidemia model mice.

Figure 9 Representative images of HE staining of aortic sinus after 30 days of administration of plasminogen in a 16-week hyperlipidemia model mouse. A and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group. The results showed that foam cell deposition (marked by the arrow) was observed in the aortic wall of the vehicle control group, and plaque deposition was severe; only mild foam cell deposition was observed in the aortic wall of the plasminogen group, and under the intima No obvious atheroma deposits were found, and the aorta in the plasminogen group was less damaged. This indicates that plasminogen can ameliorate the damage caused by lipid deposition in the aortic sinus wall of mice with hyperlipidemia.

Figure 10. Immunofibrotic staining of cardiac fibrin after 30 days of administration of plasminogen in a 16-week hyperlipidemia model mouse. A is the control vehicle PBS control group, B is the plasminogen group, and C is the quantitative analysis result. The results showed that the positive expression of cardiac fibrin in the plasminogen group was significantly less than that in the vehicle control group, and the statistical difference was significant (* indicates P<0.05). This indicates that plasminogen can reduce heart damage caused by hyperlipidemia.

Figure 11 Representative images of cardiac IgM immunostaining 30 days after administration of plasminogen in a 16-week hyperlipidemia model mouse. A is the vehicle PBS control group and B is the plasminogen group. The results showed that the positive expression of IgM in the plasminogen group was significantly less than that in the vehicle PBS control group, indicating that plasminogen can alleviate the heart damage caused by hyperlipidemia.

Figure 12 Representative pictures of Sirius red staining after 30 days of plasminogen administration in a 16-week hyperlipidemia model mouse. A is the vehicle PBS control group and B is the plasminogen group. The results showed that the deposition of collagen in the plasminogen group was significantly less than that in the vehicle PBS control group, indicating that plasminogen can alleviate cardiac fibrosis in hyperlipidemia model mice.

Figure 13 Serum troponin assay results after administration of plasminogen for 30 days in a 16-week hyperlipidemia model mouse. The results showed that the concentration of serum cardiac troponin in the control group was significantly higher than that in the plasminogen group, and the statistical difference was significant (* indicates P<0.05). This indicates that plasminogen can significantly repair the damage of hyperlipidemic heart.

Figure 14 Serum high-density lipoprotein cholesterol test results after 10 days and 20 days of plasminogen administration in 3% cholesterol hyperlipidemia model mice. The results showed that the serum HDL-C concentration in the plasminogen group was significantly higher than that in the vehicle-treated PBS group after administration of plasminogen, and the high-density lipoprotein concentrations were statistically different after 10 and 20 days of administration. Extremely significant (** means P < 0.01). It is indicated that plasminogen can effectively increase the content of high-density lipoprotein cholesterol in serum of hyperlipidemia model mice and improve dyslipidemia in hyperlipidemia model mice.

Figure 15 Results of serum total cholesterol after 20 days of administration of plasminogen in 3% cholesterol hyperlipidemia model mice. The results showed that the total cholesterol concentration in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P<0.05). This indicates that plasminogen can reduce the total cholesterol content in the serum of hyperlipidemia model mice and has the function of lowering blood lipids.

Figure 16 Serum low-density lipoprotein cholesterol test results after 20 days of plasminogen administration in 3% cholesterol hyperlipidemia model mice. The results showed that the concentration of LDL-C in the plasminogen group was significantly lower than that in the vehicle-treated PBS control group, and the statistical difference was significant (* indicates P<0.05). This indicates that plasminogen can reduce the content of low-density lipoprotein cholesterol in the serum of hyperlipidemia model mice and has the function of improving hyperlipemia.

Figure 17 Serum cardiac risk risk index results after 20 days of plasminogen administration in 3% cholesterol hyperlipidemia model mice. The results showed that the CRI for the plasminogen group was significantly smaller than that of the vehicle control group, and the statistical difference was extremely significant (** indicates P<0.01). This indicates that plasminogen can effectively reduce the risk of heart disease in mice with hyperlipidemia.

Figure 18. Liver oil red O staining picture after 35 days of plasminogen administration in 24-25 week diabetic mice. The results showed that the lipid deposition area of the liver of the plasminogen group was significantly smaller than that of the vehicle PBS control group, and the statistical difference was significant (* indicates P<0.05). This indicates that plasminogen can reduce the deposition of fat in the liver of diabetic mice.

Figure 19 is a picture of aortic HE staining after administration of plasminogen for 23 days in 24-25 week old diabetic mice. A and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group. The results showed that there were foam cell deposition (arrow mark) on the vascular wall of the control vehicle PBS control group, the middle elastic membrane was disorderly arranged, the blood vessel wall was thickened, and the tube wall was uneven; the structure of the middle elastic membrane of the plasminogen group was regular. The shape of the wave is uniform and the thickness of the vessel wall is uniform. It indicates that injection of plasminogen has a certain repairing effect on aortic injury caused by diabetes.

Figure 20 Representative pictures of ventricular oil red O staining after 26 days of administration of plasminogen in 26-week-old diabetic mice. A is the vehicle PBS control group and B is the plasminogen group. The results showed that ventricular lipid deposition (arrow markers) was significantly less in the plasminogen group than in the vehicle PBS control group. This indicates that plasminogen can reduce ventricular lipid deposition in diabetic mice and promote the repair of ventricular injury.

Figure 21 Results of detection of high-density lipoprotein cholesterol in serum after administration of plasminogen for 35 days in 26-week-old diabetic mice. The results showed that after 35 days of continuous injection of human plasmin source in diabetic mice, the serum level of HDL-C in the plasminogen group was higher than that in the vehicle control group. And the statistical difference is significant. It indicated that injection of plasminogen could promote the increase of serum high-density lipoprotein cholesterol and improve the dyslipidemia in diabetic mice.

Figure 22 Results of detection of serum low-density lipoprotein cholesterol (LDL-C) in rabbits aged 24-25 weeks of age after administration of plasminogen for 31 days. The results showed that after 31 days of continuous injection of human plasmin source in diabetic model mice, the LDL-C content in the serum of the plasminogen group was lower than that of the control vehicle PBS control group, and the statistical difference was close (P=0.1). . This indicates that plasminogen can reduce the content of low-density lipoprotein cholesterol in the serum of diabetic mice.

Figure 23 Representative pictures of aortic sinus simulone red staining after administration of plasminogen for 16 weeks in a 16-week-old hyperlipidemia model mouse. A and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group. The results showed that the area of collagen deposition (arrow mark) in the vascular sinus of the plasminogen group was significantly smaller than that of the vehicle PBS control group, indicating that plasminogen can attenuate the aortic sinus fibrosis in hyperlipidemia model mice. Level.

Figure 24 is a graph showing cardiac coefficient statistics after 30 days of plasminogen administration in ApoE atherosclerotic model mice. The results showed that the cardiac organ coefficient of the plasminogen group was significantly lower than that of the vehicle PBS control group. This indicates that plasminogen can ameliorate cardiac compensatory hypertrophy caused by cardiac injury in ApoE atherosclerotic model mice.

Figure 25 shows the results of Sirius red staining of 3% cholesterol hyperlipidemia model mice after 30 days of administration of plasminogen. A is a blank control group, B is a vehicle control group, C is a plasminogen group, and D is a quantitative analysis result. The results showed that the collagen deposition (arrow mark) in the plasminogen group was significantly less than that in the vehicle control group, and the statistical difference was significant; the fibrosis in the plasminogen group was basically restored to normal levels. This indicates that plasminogen can effectively reduce renal fibrosis in 3% cholesterol hyperlipidemia model mice.

Figure 26 shows the results of observation of renal oil red O in mice of 3% cholesterol hyperlipidemia model 30 days after administration of plasminogen. A is a blank control group, B is a vehicle control group, C is a plasminogen group, and D is a quantitative analysis result. The results showed that the renal fat deposition (arrow mark) in the plasminogen group was significantly less than that in the vehicle control group, and the quantitative analysis was statistically significant. In addition, the lipid deposition level in the plasminogen group was compared with the blank control group. The mice are similar. This indicates that plasminogen can reduce the deposition of fat in the kidney of hyperlipidemia model mice, thereby reducing kidney damage caused by fat deposition.

Example

Example 1 Plasminogen Reduces Risk of Atherosclerosis in a Model of 3% Cholesterol Hyperlipidemia Mice

Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia ^[30 , ^31] . This model was designated as a model of 3% cholesterol hyperlipidemia. The modeled mice continued to be fed a 3% cholesterol high fat diet. 50 μl of blood was taken from each mouse three days before administration, total cholesterol (T-CHO) was detected, and two groups were randomly divided into two groups according to the total cholesterol concentration and body weight. The start of administration was recorded as the first day, and the plasminogen group mice were injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. After the 20th day of administration, the mice began to fast, fasted for 16 hours, and on the 21st day, 50 μL of blood was taken from the iliac venous plexus, and the supernatant was obtained by centrifugation. The total cholesterol content was determined by the total cholesterol test kit (Nanjing Jianshe Bioengineering Research Institute). , Item No. A111-1) was tested; high-density lipoprotein cholesterol (HDL-C) content was detected using a high-density lipoprotein cholesterol test kit (Nanjing Institute of Bioengineering, item number A112-1).

The atherosclerosis index is a comprehensive indicator of clinical prediction of atherosclerosis. It is considered to be clinically more meaningful than the individual cholesterol, triglyceride, high-density lipoprotein and low-density lipids in estimating the degree of risk of coronary heart disease. The protein is larger ^[38] . Atherosclerosis index = (T-CHO-HDL-C) / HDL-C.

The calculated results showed that the atherosclerosis index of the mice in the plasminogen group was significantly lower than that in the vehicle control group, and the statistical difference was significant (Fig. 1). This indicates that plasminogen can reduce the risk of atherosclerosis in mice with hyperlipidemia.

Example 2 Plasminogen reduces serum total cholesterol in ApoE atherosclerotic mice

Thirteen male ApoE mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model ^[40,41] . Mice after modeling continue to feed high-fat, high-cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. . The first dose was started on the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. On the 30th day, the mice were fasted for 16 hours. On the 31st day, the eyeballs were removed and blood was taken. The supernatant was centrifuged to obtain a total cholesterol test using a total cholesterol test kit (Nanjing Institute of Bioengineering, item number A111-1).

The results showed that the total cholesterol concentration in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (P=0.014) (Fig. 2). This indicates that plasminogen can reduce the total cholesterol in the serum of ApoE atherosclerotic model mice and improve the dyslipidemia of atherosclerosis.

Example 3 Plasminogen reduces serum triglyceride levels in ApoE atherosclerotic mice

Thirteen male ApoE mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model ^[40,41] . Mice after modeling continue to feed high-fat, high-cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. . The first dose was recorded as the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. On the 30th day, the mice were fasted for 16 hours. On the 31st day, the eyeballs were taken for blood collection, and the supernatant was centrifuged to obtain a triglyceride detection kit (Nanjing Institute of Bioengineering, article number A110-1) for triglyceride detection.

The test results showed that the concentration of triglyceride in the plasminogen group was significantly lower than that in the control vehicle PBS control group, and the statistical difference was significant (P=0.013) (Fig. 3). This indicates that plasminogen can reduce the serum triglyceride content of ApoE atherosclerosis model mice and improve atherosclerotic dyslipidemia.

Example 4 plasminogen reduces serum low density lipoprotein cholesterol in ApoE atherosclerotic mice

Thirteen male ApoE mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model ^[40,41] . Mice after modeling continue to feed high-fat, high-cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. . The first dose was recorded as the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. On the 30th day, the mice were fasted for 16 hours. On the 31st day, the eyeballs were taken for blood collection, and the supernatant was centrifuged to obtain a low-density lipoprotein cholesterol (LDL-C) test kit (Nanjing Institute of Bioengineering, item number A113-1). LDL-C detection.

The results showed that the concentration of LDL-C in the plasminogen group was significantly lower than that in the vehicle-treated PBS control group, and the statistical difference was significant (P=0.017) (Fig. 4). Explain that plasminogen can reduce ApoE atherosclerosis Low-density lipoprotein cholesterol in the serum of the sclerotic model mice improves dyslipidemia in atherosclerotic model mice.

Example 5 Plasminogen improves liver lipid deposition in ApoE atherosclerotic mice

Thirteen male ApoE mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model ^[40,41] . Mice after modeling continue to feed high-fat, high-cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. . The first dose was recorded as the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. The mice were sacrificed on the 31st day, and the liver tissues were fixed in 4% paraformaldehyde for 24-48 hours, respectively, in a 15%, 30% sucrose solution at 4 ° C overnight, embedded in OCT, frozen section thickness 8 μm, oil red O Dyeing for 15 min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30s, glycerin gelatin for sealing. Sections were observed under a 400x optical microscope.

The staining results showed that the liver fat deposition in the plasminogen group (Fig. 5B) was significantly less than that in the vehicle PBS control group (Fig. 5A), and the statistical analysis was statistically significant (P = 0.02) (Fig. 5C). This indicates that plasminogen can reduce the deposition of fat in the liver of atherosclerotic model mice.

Example 6 Plasminogen improves lipid deposition in aortic sinus of ApoE atherosclerotic mice

Thirteen male ApoE mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model ^[40,41] . Mice after modeling continue to feed high-fat, high-cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. . The first dose was recorded as the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. The mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours. The cells were sedimented in 15% and 30% sucrose at 4 °C overnight, embedded in OCT, and the frozen section was 8 μm thick. Oil red O Dyeing for 15 min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30s, glycerin gelatin for sealing. Sections were observed under a 40x optical microscope.

The staining results showed that the aortic sinus fat deposition in the plasminogen group (Fig. 6B) was significantly less than that in the vehicle PBS control group (Fig. 6A). This indicates that plasminogen can reduce the deposition of lipids in the aortic sinus of atherosclerotic model mice.

Example 7 Degradation of plasminogen in the liver of a 16-week hyperlipidemia model mouse

11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model ^[30,31] . This model was assigned to 16-week hyperlipidemia. model. Mice after modeling continue to feed high cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. . The start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. The mice were sacrificed for 30 days, and the mice were sacrificed on the 31st day. The liver was fixed in 4% paraformaldehyde for 24-48 hours, respectively, in a 15%, 30% sucrose solution at 4 ° C overnight, embedded in OCT, and frozen section thickness. 8μm, oil red O staining for 15min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30s, glycerin gelatin sealing. Sections were observed under a 400x optical microscope.

Oil red O staining can show lipid deposition, reflecting the extent of lipid deposition ^[32] . The results showed that the liver fat deposition in the plasminogen group (Fig. 7B) was significantly less than that in the vehicle PBS control group (Fig. 7A), and the statistical analysis was statistically significant (Fig. 7C). This indicates that plasminogen can reduce the deposition of fat in the liver of hyperlipidemia model mice.

Example 8 Plasminogen-reducing lipid deposition in aortic sinus of a 16-week hyperlipidemia model mouse

11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model ^[30,31] . This model was assigned to 16-week hyperlipidemia. model. Mice after modeling continue to feed high cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. . The start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. The mice were sacrificed for 30 days, and the mice were sacrificed on the 31st day. The heart tissue was fixed in 4% paraformaldehyde for 24-48 hours, respectively, in a 15%, 30% sucrose overnight at 4 ° C, OCT embedding, aorta The sinus frozen section thickness was 8 μm, oil red O staining for 15 min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30 s, and glycerin gelatin for sealing. The sections were observed under 40 (Fig. 8A, 8B), 200 times (Fig. 8C, 8D) magnification optical microscope.

The results showed that the aortic sinus fat deposition in the plasminogen group (Fig. 8B, 8D) was significantly less than that in the vehicle PBS control group (Fig. 8A, 8C), and the statistical difference was significant (Fig. 8E). This indicates that plasminogen can reduce the deposition of lipids in the aortic sinus of hyperlipidemia model mice.

Example 9 Plasminogen Amelioration of Aortic Sinus Injury in a Model of Hyperlipidemia in 16 Weeks

11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model ^[30,31] . This model was assigned to 16-week hyperlipidemia. model. Mice after modeling continue to feed high cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. . The start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. The mice were administered for 30 days, and the mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene. The fixed tissue samples were dehydrated by alcohol gradient and transparent to xylene for paraffin embedding. The thickness of the aortic sinus tissue section was 3 μm. The sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining). After 1% hydrochloric acid alcohol was differentiated, the ammonia water returned to blue and dehydrated with an alcohol gradient. The slices were at 40 (Fig. 9A, 9B). 200 times (Fig. 9C, 9D) observed under an optical microscope.

The results showed that foam cells were deposited on the inner wall of the aortic sinus in the vehicle PBS control group (Fig. 9A, 9C), and the plaque was deposited heavily; only the inner wall of the aortic sinus was visible in the plasminogen group (Fig. 9B, 9D). Mild foam cell deposition, and no obvious atheroma deposits under the intima, less damage to the inner wall of the aortic sinus in the plasminogen group. This indicates that plasminogen can improve the damage of the sinus wall of mice with hyperlipidemia.

Example 10 Plasminogen reduces the expression of cardiac fibrin in a mouse model of hyperlipidemia at 16 weeks

11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model ^[30,31] . This model was assigned to 16-week hyperlipidemia. model. Mice after modeling continue to feed high cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. . The start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. The mice were administered for 30 days, and the mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene. The thickness of the tissue section was 3 μm, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 5% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; after the time, the sheep serum was discarded and the tissue was circled with a PAP pen. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. Rabbit anti-mouse fibrin antibody (Abeam) was incubated overnight at 4 ° C and washed twice with 0.01 M PBS for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and the neutral gum was sealed, and the sections were observed under a 200-fold optical microscope.

Fibrinogen is a precursor of fibrin. In the case of tissue damage, as a stress response to the body, fibrinogen is hydrolyzed into fibrin deposits at the site of injury ^[33,34] . Therefore, the level of damaged local fibrin can be used as a marker of the degree of damage.

Immunohistochemical staining showed that the positive expression of cardiac fibrin in the plasminogen group (Fig. 10B) was significantly less than that in the vehicle PBS control group (Fig. 10A), and the statistical difference was significant (Fig. 10C), indicating that fibrinolysis The zymogen can reduce myocardial damage caused by hyperlipidemia.

Example 11 Plasminogen effectively protects myocardial injury in a 16-week hyperlipidemia model mouse

11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model ^[30,31] . This model was assigned to 16-week hyperlipidemia. model. Mice after modeling continue to feed high cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. . The start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. The mice were administered for 30 days, and the mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene. The thickness of the tissue section was 3 μm, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 5% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; after the time, the sheep serum was discarded and the tissue was circled with a PAP pen. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. Goat anti-mouse IgM (HRP) antibody (Abeam) was incubated for 1 hour at room temperature and washed twice with PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and the hematoxylin was stained with nuclei for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent, and the neutral gum was sealed, and the sections were observed under a 200-fold optical microscope.

IgM antibodies play an important role in the clearance of apoptotic and necrotic cells, and the level of local IgM antibodies in damaged tissues is positively correlated with the degree of injury ^[35,36] . Therefore, detecting the level of local IgM antibodies in tissues and organs can reflect the degree of damage of the tissues and organs.

Immunostaining results showed that the positive expression of cardiac IgM in the plasminogen group (Fig. 11B) was significantly less than that in the vehicle PBS control group (Fig. 11A), indicating that plasminogen can reduce the heart of hyperlipidemia model animals. damage.

Example 12 Plasminogen attenuates cardiac fibrosis in a 16-week hyperlipidemia model mouse

11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model ^[30,31] . This model was assigned to 16-week hyperlipidemia. model. Mice after modeling continue to feed high cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. . The start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. The mice were administered for 30 days, and the mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene. The thickness of the tissue section was 3 μm. The sections were dewaxed and rehydrated, washed once with water, stained with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, stained with hematoxylin for 1 minute, rinsed with water, differentiated with 1% hydrochloric acid, and returned to blue with ammonia. Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.

Sirius red staining can make collagen staining for a long time. As a special staining method for pathological sections, Sirius red staining can specifically display collagen tissue.

The staining results showed that the deposition of collagen in the plasminogen group (Fig. 12B) was significantly less than that in the vehicle PBS control group (Fig. 12A), indicating that plasminogen can alleviate the deposition of collagen in the heart tissue of hyperlipidemia model mice. To reduce myocardial fibrosis.

Example 13 plasminogen repair 16-week hyperlipidemia model mice myocardial injury

11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model ^[30,31] . This model was assigned to 16-week hyperlipidemia. model. Mice after modeling continue to feed high cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. . The start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. After 30 days of administration, the mice began to fast after the 30th day of administration, fasting for 16 hours. On the 31st day, the eyeballs were taken for blood collection, and the supernatant was obtained by centrifugation. Cardiac troponin I (CTNI) detection kit was used. (Nanjing built) to measure the concentration of troponin in serum.

Cardiac troponin I is an important marker of myocardial injury, and its serum concentration can reflect the extent of myocardial damage ^[37] .

The test results showed that the serum troponin concentration in the control vehicle PBS control group was significantly higher than that in the plasminogen group, and the statistical difference was significant (Fig. 13). This indicates that plasminogen can significantly improve cardiac damage in hyperlipidemia model mice.

Example 14 Plasminogen Increases Serum High Density Lipoprotein Cholesterol Concentration in 3% Cholesterol Hyperlipidemia Model Mice

Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia ^[30 , ^31] . This model was designated as a model of 3% cholesterol hyperlipidemia. The modeled mice continued to be fed a 3% cholesterol high fat diet. 50 μL of blood was taken from each mouse three days before the administration, and total cholesterol was measured, and was randomly divided into two groups according to the total cholesterol concentration and body weight, with 8 in each group. The first dose was recorded as the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 20 days. On the 10th and 20th day, the mice were fasted for 16 hours. On the 11th and 21st day, 50 μl of blood was taken from the iliac venous plexus, and the supernatant was centrifuged to detect serum high-density lipoprotein cholesterol (HDL-C). The high-density lipoprotein cholesterol content in this paper was detected by the method described in the test kit (Nanjing Institute of Bioengineering, item number A112-1).

High-density lipoprotein is an anti -atherosclerotic plasma lipoprotein , a protective factor for coronary heart disease, commonly known as "vascular scavenger."

The results showed that the serum HDL-C concentration in the plasminogen group was significantly higher than that in the vehicle PBS control group, and the HDL-C concentrations were statistically different after 10 and 20 days of administration (Fig. 14). It is indicated that plasminogen can increase the content of high-density lipoprotein cholesterol in serum of hyperlipidemia model mice and improve dyslipidemia in hyperlipidemia mice.

Example 15 Plasminogen Reduces Serum Total Cholesterol Level in 3% Cholesterol Hyperlipidemia Model Mice

Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia ^[30 , ^31] . This model was designated as a model of 3% cholesterol hyperlipidemia. The modeled mice continued to be fed a 3% cholesterol high fat diet. 50 μL of blood was taken from each mouse three days before the administration, and total cholesterol was measured, and was randomly divided into two groups according to the total cholesterol concentration and body weight, with 8 in each group. The first dose was recorded as the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 20 days. On the 20th day, the mice were fasted for 16 hours. On the 21st day, 50 μL of blood was taken from the iliac venous plexus, and the supernatant was obtained by centrifugation. Total cholesterol test was performed using the total cholesterol test kit (Nanjing Institute of Bioengineering, article number A111-1). .

The results showed that the total cholesterol concentration in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (Fig. 15). This indicates that plasminogen can reduce the total cholesterol in the serum of hyperlipidemia model mice.

Example 16 Plasminogen Reduces Serum Low Density Lipoprotein Cholesterol Level in 3% Cholesterol Hyperlipidemia Model Mice

Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia ^[30 , ^31] . This model was designated as a model of 3% cholesterol hyperlipidemia. The modeled mice continued to be fed a 3% cholesterol high fat diet. 50 μl of blood was taken from each mouse three days before the administration, and total cholesterol was measured, and was randomly divided into two groups according to the total cholesterol concentration and body weight, with 8 in each group. The first dose was recorded as the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 20 days. On the 20th day, the mice were fasted for 16 hours. On the 21st day, 50 μL of blood was taken from the iliac venous plexus, and the supernatant was obtained by centrifugation. The low-density lipoprotein cholesterol test kit (Nanjing Institute of Bioengineering, item number A113-1) was used. Low density lipoprotein cholesterol (LDL-C) detection.

Low-density lipoprotein is a lipoprotein particle that carries cholesterol into peripheral tissue cells and can be oxidized to oxidized low-density lipoprotein when low-density lipoproteins, especially oxidized low-density lipoprotein (OX-LDL), are excessive. The cholesterol it carries will accumulate on the arterial wall and cause arteriosclerosis. Therefore, low density lipoprotein cholesterol is called "bad cholesterol."

The results showed that the concentration of the plasminogen group (LDL-C) was significantly lower than that of the vehicle PBS control group, and the statistical difference was significant (Fig. 16). It is indicated that plasminogen can reduce the content of low-density lipoprotein cholesterol in serum of hyperlipidemia model mice and improve dyslipidemia in hyperlipidemia mice.

Example 17 Plasminogen Reduces Cardiac Risk in 3% Cholesterol Hyperlipidemia Model Mice

Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia ^[30 , ^31] . This model was designated as a model of 3% cholesterol hyperlipidemia. The modeled mice continued to be fed a 3% cholesterol high fat diet. 50 μl of blood was taken from each mouse three days before administration, and total cholesterol (T-CHO) was measured, and randomly divided into two groups according to the total cholesterol concentration, 8 in each group. The start of administration was recorded as the first day, and the plasminogen group mice were injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. After the 20th day of administration, the mice began to fast, fasted for 16 hours, and on the 21st day, 50 μL of blood was taken from the iliac venous plexus, and the supernatant was obtained by centrifugation. The total cholesterol content was determined by the total cholesterol test kit (Nanjing Jianshe Bioengineering Research Institute). , Item No. A111-1) was tested; high-density lipoprotein cholesterol (HDL-C) content was detected using a high-density lipoprotein cholesterol test kit (Nanjing Institute of Bioengineering, item number A112-1). Cardiac risk index = T-CHO/HDL-C.

Cardiac risk index (CRI) is used to assess the risk of dyslipidemia-induced heart disease ^[38] .

The results showed that the CRI for the plasminogen group was significantly smaller than that for the vehicle PBS control group, and the statistical difference was extremely significant (Fig. 17). This indicates that plasminogen can effectively reduce the risk of heart disease in mice with hyperlipidemia.

Example 18 Plasminogen Improves Lipid Deposition in Liver of Diabetic Mice

Ten male db/db mice aged 24-25 weeks were recorded as day 0 on the day of the experiment and weighed. They were randomly divided into two groups according to body weight, and 5 rats in the vehicle PBS control group and the plasminogen group. Start with plasminogen or PBS on the 1st day. The plasminogen group was injected with plasminogen at a dose of 2 mg/0.2 ml/day/day, and the same volume of PBS was administered to the vehicle PBS control group for 30 days. On the 36th day, the mice were sacrificed and the liver tissue was fixed with 4% paraformaldehyde for 24-48 hours. They were sacrificed in 15% and 30% sucrose at 4 °C overnight, embedded in OCT, frozen sections were 8 μm thick, and oil red O stained for 15 min. 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30s, glycerin gelatin for sealing. Sections were observed under a 200x optical microscope.

The staining results showed that the lipid deposition area of the liver of the plasminogen group (Fig. 18B) was significantly smaller than that of the vehicle PBS control group (Fig. 18A), and the statistical difference was significant (P = 0.02) (Fig. 18C). This indicates that plasminogen can reduce the deposition of fat in the liver of diabetic mice.

Example 19 Plasminogen Attenuates Aortic Wall Damage in Diabetic Mice

Ten male db/db mice aged 24-25 weeks were recorded as day 0 on the day of the experiment and weighed. They were randomly divided into two groups according to body weight, and 5 rats in the vehicle PBS control group and the plasminogen group. PBS or plasminogen was administered to the first day for 31 days. The plasminogen group was injected with plasminogen at a dose of 2 mg/0.2 ml/day/day, and the same volume of PBS was administered to the vehicle PBS control group by tail vein injection. Mice were sacrificed on day 32 and the aorta was fixed in 10% neutral formalin fixative for 24 hours. The fixed aorta was paraffin-embedded by alcohol gradient dehydration and xylene transparency. Tissue section thickness 5 μm, section dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), 1% salt After the acid alcohol was differentiated, the ammonia water returned to the blue and was dehydrated and sealed with an alcohol gradient. The sections were observed under a 400-fold (Fig. 19A, 19B) optical microscope and a 1000-fold (Fig. 19C, 19D) oil microscope.

Diabetes with hyperlipidemia is a common complication of diabetes and an important risk factor for diabetic macroangiopathy ^[39] .

The staining results showed that there was foam cell deposition (arrow mark) on the vessel wall of the vehicle PBS control group (Fig. 19A, 19C), the middle layer elastic membrane was disorderly arranged, the vessel wall was thickened, and the tube wall was uneven; the plasminogen group was given. (Fig. 19B, 19D) The structure of the middle elastic membrane is regular, wavy, and the thickness of the vessel wall is uniform. It indicates that injection of plasminogen can alleviate the lipid deposition in the aortic wall of diabetic mice, and has a certain protective effect on the damage caused by lipid deposition in arterial wall.

Example 20 Plasminogen Reduces Ventricular Lipid Deposition in Diabetic Mice

Nineteen male db/db mice at 26 weeks of age were randomly assigned to 4 rats in the plasminogen group and 5 to the vehicle PBS control group. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 35 days. The mice were sacrificed on the 36th day, and the hearts were fixed in 4% paraformaldehyde for 24-48 hours, respectively, in 15%, 30% sucrose at 4 ° C overnight, embedded in OCT, frozen section thickness 8 μm, oil red O staining 15 min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30s, glycerin gelatin seal. Sections were observed under a 200x optical microscope.

The results showed that ventricular lipid deposition (arrow mark) was significantly less in the plasminogen group (Fig. 20B) than in the vehicle PBS control group (Fig. 20A). This indicates that plasminogen can reduce the deposition of fat in the ventricle of diabetic mice and promote the repair of ventricular damage.

Example 21 Plasminogen Increases High Density Lipoprotein Cholesterol Levels in Serum of Diabetic Mice

Twenty male 26-week-old db/db mice were recorded on day 0 as the day of the experiment and weighed. They were randomly divided into two groups according to body weight, 11 to the plasminogen group and 9 to the vehicle PBS control group. On day 1, plasminogen or PBS was administered continuously for 35 days. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail vein of the control group. On the 36th day, the mice were harvested from the eyeballs for whole blood, centrifuged at 3500C/min for 10 minutes at 4°C, and the supernatant was taken and high-density in serum was detected using the High Density Lipoprotein Test Kit (Nanjing Institute of Bioengineering, Cat. No. A112-1). Lipoprotein cholesterol (HDL-C) concentration.

The results showed that the serum levels of HDL-C in the plasminogen group were higher than those in the vehicle-treated PBS control group (Fig. 21). It indicates that injection of plasminogen can promote the increase of serum high-density lipoprotein cholesterol and improve the dyslipidemia of diabetes.

Example 22 Plasminogen Reduces Low Density Lipoprotein Cholesterol in Serum of Diabetic Mice

Ten male db/db mice aged 24-25 weeks were recorded as day 0 on the day of the experiment and weighed. They were randomly divided into two groups according to body weight, and 5 rats in the plasminogen group and the vehicle control PBS control group, respectively. Three db/m were taken as normal control groups. On day 1, plasminogen or PBS was administered continuously for 31 days. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail vein of the PBS control group. The normal control mice were not treated. On the 32nd day, the mice were harvested from the eyeballs for whole blood, centrifuged at 3500C/min for 10 minutes at 4°C, and the supernatant was taken and tested in serum using the Low Density Lipoprotein Cholesterol Detection Kit (Nanjing Institute of Bioengineering, Cat. No. A113-1). Low density lipoprotein cholesterol (LDL-C) concentration.

The results showed that after 31 days of continuous injection of human plasmin source in diabetic model mice, the LDL-C content in the serum of the plasminogen group was lower than that of the vehicle control group, and the statistical difference was close (P=0.1). ) (Figure 22). This indicates that plasminogen can reduce the content of LDL-C in serum.

Example 23 Plasminogen Reduction of Aortic Sinus Fibrosis in a Model of Hyperlipidemia in 16 Weeks

11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model ^[30,31] . This model was assigned to 16-week hyperlipidemia. model. Mice after modeling continue to feed high cholesterol feed. 50 μl of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. . The start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. After 30 days of administration, the mice were sacrificed on the 31st day, and the hearts were fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene. The thickness of the aortic sinus slice was 3μm. The slice was dewaxed and rehydrated and washed once. After staining with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 minutes, hematoxylin staining for 1 minute, running water rinse, 1% hydrochloric acid alcohol differentiation, ammonia water. Return to blue, rinse with water, and dry the neutral gum seal, and observe under 40 (Fig. 23A, 23B), 200 times (Fig. 23C, 23D) light microscope.

The results showed that the area of collagen deposition (arrow mark) in the aortic sinus vascular wall of the plasminogen group (Fig. 23B, 23D) was significantly smaller than that of the vehicle PBS control group (Fig. 23A, 23C), indicating that plasminogen can be reduced. Aortic sinus fibrosis levels in hyperlipidemia model mice.

Example 24 Plasminogen Improves Compensatory Hypertrophy in ApoE Atherosclerotic Mice

Thirteen male ApoE mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model ^[40,41] . The mice after the modeling were taken 50 μl of blood for three days before the administration to detect the total cholesterol (T-CHO) content, and were randomly divided into two groups according to the T-CHO content, and the vehicle PBS control group was given 7 cells. 6 lysogen group. The first dose was started on the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. After 30 days of administration, the high fat and high cholesterol feed was continued to be fed during the administration. The mice were sacrificed on the 31st day after the administration, the hearts were weighed, and the cardiac coefficient was calculated. Cardiac coefficient (%) = heart weight / body weight × 100.

The results showed that the cardiac coefficient of the plasminogen group was significantly lower than that of the vehicle PBS control group (Fig. 24). This indicates that plasminogen can attenuate cardiac compensatory hypertrophy caused by cardiac injury in ApoE atherosclerotic model mice.

Example 25 Plasminogen Reduces Kidney Fibrosis in a Model of 3% Cholesterol Hyperlipidemia Mice

Sixteen male C57 mice of 9 weeks old were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia ^[30 , ^31] . This model was classified as a model of 3% cholesterol hyperlipidemia. The modeled mice continued to be fed a 3% cholesterol high fat diet. Another 5 male C57 mice of the same age were used as a blank control group, and normal maintenance feed was fed during the experiment. Three days before the administration, 50 μL of blood was taken from each mouse to measure total cholesterol. The model mice were randomly divided into two groups according to the total cholesterol concentration and body weight, and given to the plasminogen group and the vehicle PBS control group, each group was 8 only. The start of administration was recorded as the first day, and the plasminogen group mice were injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. The drug was administered for 30 days, and the mice were sacrificed on the 31st day, and the kidneys were fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene. The thickness of the kidney tissue section was 3 μm. The sections were dewaxed and rehydrated, washed once with water, stained with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, stained with hematoxylin for 1 minute, rinsed with water, differentiated with 1% hydrochloric acid, and returned with ammonia. Blue, rinsed with water, dried and then sealed with a neutral gum, observed under a 200x optical microscope.

The results showed that kidney collagen deposition (arrow mark) was significantly less in the plasminogen group (Fig. 25C) than in the vehicle PBS control group (Fig. 25B), and the statistical difference was significant (Fig. 25D); plasminogen group fibers were given. Basically returned to normal levels (Figure 25A). This indicates that plasminogen can effectively reduce renal fibrosis in 3% cholesterol hyperlipidemia model mice.

Example 26 Plasminogen Reduces Renal Fat Deposition in 3% Cholesterol Hyperlipidemia Model Mice

Sixteen male C57 mice of 9 weeks old were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia ^[30 , ^31] . This model was classified as a model of 3% cholesterol hyperlipidemia. The modeled mice continued to be fed a 3% cholesterol high fat diet. Another 5 male C57 mice of the same age were used as a blank control group, and normal maintenance feed was fed during the experiment. Three days before the administration, 50 μL of blood was taken from each mouse to measure total cholesterol. The model mice were randomly divided into two groups according to the total cholesterol concentration and body weight, and given to the plasminogen group and the vehicle PBS control group, each group was 8 only. The first dose was recorded as the first day. The plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. On the 31st day, the mice were sacrificed and the kidneys were fixed in 4% paraformaldehyde for 24-48 hours. They were sedimented in 15% and 30% sucrose at 4 °C overnight, embedded in OCT, frozen sections were 8 μm thick, and oil red O stained for 15 min. 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30 seconds, glycerin gelatin seal. Sections were observed under a 400x optical microscope.

The results showed that the kidney fat deposition (arrow mark) in the plasminogen group (Fig. 26C) was significantly less than that in the vehicle PBS control group (Fig. 26B), and the quantitative analysis was statistically significant (Fig. 26D); Lipid deposition levels in the lysogen group were similar to those in the blank control group (Fig. 26A). It is indicated that plasminogen can reduce the deposition of fat in the kidney of hyperlipidemic model mice, thereby reducing the damage of kidney caused by fat deposition.

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Claims (47)

1. A method of preventing atherosclerosis comprising administering a prophylactically effective amount of plasminogen to a subject susceptible to atherosclerosis.
2. The method of claim 1, wherein said subject susceptible to atherosclerosis is susceptible or suffering from a disorder of fat metabolism, a disease of glucose metabolism, a liver disease, a kidney disease, a cardiovascular disease, an intestinal disease, a thyroid disease , gallbladder or biliary tract disease, subjects with obesity, or subjects who drink excessively.
3. The use according to claim 2, wherein said subject susceptible to atherosclerosis is susceptible or suffering from hypertension, diabetes, chronic hepatitis, cirrhosis, kidney injury, chronic glomerulonephritis, chronic pyelonephritis, Nephrotic syndrome, renal insufficiency, kidney transplantation, uremia, hypothyroidism, obstructive cholecystitis, obstructive cholangitis.
4. The method of claim 1 wherein said subject susceptible to atherosclerosis is a subject having congenital or secondary obesity.
5. The method according to any one of claims 1 to 4, wherein the subject susceptible to atherosclerosis is susceptible or suffering from hypertension, diabetes, hyperlipidemia, hyperlipoproteinemia or fatty liver. Subject.
6. A method of preventing and/or reducing abnormal deposition of fat in a body tissue of a subject, comprising administering to the subject an effective amount of plasminogen.
7. The method of claim 6 wherein said abnormal deposition of fat in body tissues and organs refers to abnormal deposition of fat in blood, subcutaneous tissue, blood vessel walls, and internal organs.
8. A method of preventing or treating a disease in a subject by attenuating the abnormal deposition of fat in body tissues and organs, comprising administering to the subject an effective amount of plasminogen.
9. The method of claim 8 wherein said disease comprises obesity, fatty liver, cirrhosis, atherosclerosis, coronary heart disease.
10. A method of preventing atherosclerosis in a subject with hyperlipidemia comprising administering to the subject an effective amount of plasminogen.
11. The method of claim 10 wherein said subject has one or more of a blood lipid: elevated serum total cholesterol levels, elevated serum triglyceride levels, and elevated serum low density lipoprotein levels.
12. The method of claim 10 or 11, wherein said plasminogen prevents atherosclerosis by one or more of the following: lowering serum total cholesterol levels in the subject, lowering serum triglyceride levels in the subject, and reducing the test Serum low-density lipoprotein levels increase serum high-density lipoprotein levels in subjects.
13. A method of preventing atherosclerosis comprising administering a plasminogen to a subject to prevent atherosclerosis by one or more selected from the group consisting of: promoting fat metabolism in the liver, promoting fat transport in the liver, and Reduces fat deposition in the liver of the subject.
14. The method of claim 13 further comprising preventing atherosclerosis by one or more of: lowering serum total cholesterol levels in the subject, lowering serum triglyceride levels in the subject, and lowering serum low density lipoprotein levels in the subject Increase serum high-density lipoprotein levels in subjects.
15. The method of claim 13 or 14, further comprising preventing atherosclerosis by reducing deposition of fat in the vessel wall.
16. A method of preventing coronary atherosclerosis and/or coronary heart disease in a subject comprising administering to the subject an effective amount of plasminogen.
17. The method of claim 16, wherein the plasminogen prevents coronary atherosclerosis and/or coronary heart disease in a subject by one or more selected from the group consisting of promoting liver fat metabolism, promoting liver fat transport, and reducing Fat deposition in the liver of the subject.
18. The method of claim 16 or 17, wherein the plasminogen prevents coronary atherosclerosis and/or coronary heart disease in a subject by one or more selected from the group consisting of: lowering serum total cholesterol levels in the subject, and reducing the test Serum triglyceride levels, lower serum low-density lipoprotein levels in subjects, and elevated serum high-density lipoprotein levels in subjects.
19. The method of any one of claims 16-18, wherein the plasminogen prevents coronary atherosclerosis and/or coronary heart disease in a subject by decomposing deposition of fat in the aortic and/or coronary vessel wall.
20. A method of preventing an atherosclerosis-related disorder comprising administering an effective amount of plasminogen to a subject susceptible to atherosclerosis.
21. 20. The method of claim 20, wherein the related condition is a condition caused by arterial stenosis or arterial thrombosis due to atherosclerosis.
22. The method of claim 21, wherein the condition is one or more selected from the group consisting of coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, heart failure, cerebral ischemia, cerebral thrombosis, carotid thrombosis, brain atrophy, brain Bleeding, cerebral embolism, renal insufficiency, hypertension, glomerular fibrosis, renal failure, uremia, intestinal necrosis, blood in the stool, intermittent claudication, gangrene.
23. A method of treating hyperlipidemia comprising administering to a subject an effective amount of plasminogen.
24. The method of claim 23, wherein said hyperlipidemia is one or more selected from the group consisting of elevated serum total cholesterol levels, elevated serum triglyceride levels, and elevated serum low density lipoprotein levels.
25. A method of preventing atherosclerosis in a diabetic patient comprising administering an effective amount of plasminogen to a diabetic patient.
26. A method of preventing fatty liver comprising administering to a subject an effective amount of plasminogen.
27. The use according to any one of claims 1 to 26, wherein the plasminogen can be administered in combination with one or more other drugs and methods of treatment.
28. The method of claim 27, wherein said one or more other drugs comprise: hypolipidemic drugs, antiplatelet drugs, blood pressure lowering drugs, dilated vascular drugs, hypoglycemic drugs, anticoagulant drugs, thrombolytic drugs, liver protecting drugs , anti-arrhythmia drugs, cardiotonic drugs, diuretic drugs, anti-infective drugs, anti-viral drugs, immunomodulatory drugs, inflammatory regulatory drugs, hormone drugs.
29. The method of any one of claims 1 to 28, wherein said plasminogen and sequence 2, 6, 8, 10 or 12 have at least 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98% or 99% sequence identity and still have plasminogen activity.
30. The method according to any one of claims 1 to 29, wherein the plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 1-100, 1-90, 1- 80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, A protein of 1-4, 1-3, 1-2, 1 amino acid, and still having plasminogen activity.
31. The method of any one of claims 1 to 30, wherein the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
32. The method according to any one of claims 1 to 31, wherein the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, and delta-plasmin Pro- or their variants that retain plasminogen activity.
33. The method of any of claims 1-32, wherein the plasminogen is a natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
34. The method of any one of claims 1 to 32, wherein the plasminogen is a human plasminogen ortholog from a primate or a rodent or a variant thereof that still retains plasminogen activity or Fragment.
35. The method of any one of claims 1 to 34, wherein the amino acid of plasminogen is as shown in sequence 2, 6, 8, 10 or 12.
36. The method of any one of claims 1 to 35, wherein the plasminogen is human natural plasminogen.
37. The method of any of claims 1-36, wherein the subject is a human.
38. The method of any of claims 1-37, wherein the subject lacks or lacks plasminogen.
39. 38. The method of claim 38, wherein the deficiency or deficiency is innate, secondary, and/or local.
40. A plasminogen for use in the method of any of claims 1-39.
41. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the method of any of claims 1-39.
42. A prophylactic or therapeutic kit comprising: (i) plasminogen for use in the method of any of claims 1-39 and (ii) for delivery of said plasminogen to The subject of the subject.
43. A kit according to claim 42, wherein the member is a syringe or vial.
44. The kit of claim 42 or 43, further comprising a label or instructions for use, the label or instructions for use instructing administration of the plasminogen to the subject to perform any of claims 1-39 method.
45. An article comprising:

    a container containing a label; and

    Included in (i) a plasminogen or a plasminogen-containing pharmaceutical composition for use in the method of any of claims 1-39, wherein said label indicates that said plasminogen or composition is administered The subject is administered to perform the method of any of claims 1-39.
46. The kit of any of claims 42-44 or the article of claim 45, further comprising one or more additional members or containers containing other drugs.
47. The kit or article of claim 46, wherein said other drug is selected from the group consisting of a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, and a liver protection Drugs, antiarrhythmic drugs, cardiotonic drugs, diuretic drugs, anti-infective drugs, antiviral drugs, immunomodulatory drugs, inflammatory regulatory drugs, anti-tumor drugs, hormone drugs, thyroxine.

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